CN106754309A - It is applied to the full-automatic RNA libraries preparation facilities of second generation high-flux sequence - Google Patents

It is applied to the full-automatic RNA libraries preparation facilities of second generation high-flux sequence Download PDF

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CN106754309A
CN106754309A CN201611190715.3A CN201611190715A CN106754309A CN 106754309 A CN106754309 A CN 106754309A CN 201611190715 A CN201611190715 A CN 201611190715A CN 106754309 A CN106754309 A CN 106754309A
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reagent cabin
reagent
cabin
minutes
pipettor
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CN106754309B (en
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尚小云
李艳艳
张凯宁
靖相密
安苗苗
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Shandong Acv Biotechnologies Co Ltd
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Shandong Acv Biotechnologies Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Abstract

The present invention relates to a kind of full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence.Including box body, injection port, library transfer port are set, box body bottom is respectively provided with 1 library collecting pit, 1 sample cell, 9 agent bins, 1 waste liquid tank, device sequence measurement, step on the top surface of box body：All reagents used in RNA library constructions are all placed in advance in the agent bin of box body bottom, RNA sample through reverse transcription be cDNA, DNA fragmentation, end repair, adjunction head, PCR amplification and after purification, sampling probe is transferred to the library for building in the collecting pit of library, closure at the top of box body is opened, the library for building is taken out by library transfer port, is then just put into microarray dataset and is sequenced.Whole storehouse process of founding a capital is completed in tray interior, and whole process closing is not contacted with the external world, will not produce pollution, even one of ordinary skilled in the art can independent operation implementation, improve efficiency.

Description

It is applied to the full-automatic RNA libraries preparation facilities of second generation high-flux sequence
（One）Technical field
It is more particularly to a kind of to be applied to the full-automatic of second generation high-flux sequence the invention belongs to biomedical devices technical field RNA libraries preparation facilities.
（Two）Background technology
Used as one of most important molecular biological analysis method, the appearance of sequencing technologies is not only announcement and the base of hereditary information Because the research of the fundamental biological knowledge such as expression regulation provides significant data, and in the application study such as gene diagnosis and gene therapy Play an important role.
High throughput sequencing technologies are the milestones that development course is sequenced, and it is preceding for modern life science research is provided Not some opportunities.Second generation high throughput sequencing technologies (sequencing of two generations) can carry out gene order-checking, transcript profile sequencing, gene table Up to regulation and control, the detection of Binding site for transcription factor and the research such as methylate.The sequencing of two generations is also currently used for parsing heredity The problems such as sick genetic mechanism, pharmacogenomics, tumour personalized treatment, biomarker are identified is most widely used also most straight The technological means for connecing.
In the experimental procedure of two generations sequencing, the preparation of sequencing library is a very crucial step.Traditional sequencing library Preparation needs sample nucleic acid to extract, enzyme pre-treatment or mechanical shearing, adjunction head are chained, PCR amplifications etc. operation, and operation is multiple Miscellaneous, automaticity is not high.Simultaneously in order to avoid pollution, above operation needs to be carried out in different laboratories, to laboratory Physical space distribution has is strict with, and needs operating personnel by professional training, will otherwise influence follow-up sequencing result Accuracy.
（Three）The content of the invention
The present invention is in order to make up that prior art second generation sequencing library technology of preparing automaticity is low and easily draw in operating process Enter pollution, so that the accuracy of sequencing result is influenceed, and operation sequence specialty is cumbersome, and layman is unable to complete independently It is not enough, there is provided a kind of full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is high that this is applied to the second generation The full-automatic RNA libraries preparation facilities of flux sequencing will build the process full automation in storehouse and purifying, and realize totally closed operation, Realize no pollution, prepared by the library of high duplication, also solve the not enough problem of part Experiment room physical space.
The present invention is achieved through the following technical solutions：
A kind of full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, including box body, set on the top surface of box body Injection port is put, it is characterized in that：Also set up library transfer port, in injection port, library transfer port set can horizontally slip with Injection port, the closure of library transfer port are opened in closing, and box body bottom is respectively provided with 1 library collecting pit, 1 sample cell, 9 Individual agent bin, 1 waste liquid tank, wherein, sample cell is oppositely arranged with injection port, and library collecting pit is relative with library transfer port to be set Put, screw rod, sampling probe are respectively provided with box body, the left and right two ends of screw rod are respectively through bearing three, bearing four and box body corresponding side Connection is rotated, screw rod is horizontally disposed with, and the right-hand member of screw rod stretches out box body and is connected with screw rod knob, connector is set on screw rod, the company Fitting is connected with screw rod by screw thread, and sampling probe accommodating hole, the central shaft of sampling probe accommodating hole are provided with the top surface of connector With the central axis of screw rod, sampling probe accommodating hole bottom is fixedly installed the first spring, and the first spring leads to sampling probe accommodating hole Axle is set, and the first spring is extended by sampling probe accommodating hole bottom up and stretches out sampling probe accommodating hole, and sampling probe is set in first In spring, sampling probe is connected by pipeline with negative pressure generator, and negative pressure generator includes housing, piston and piston rod, piston rod On be cased with second spring, the free end of piston rod is fixedly connected the left end of depression bar pin, and the right-hand member of depression bar pin stretches out box body, for pushing away Enter piston rod, pressing plate is provided with above sampling probe, pressing plate includes pressing plate body and the pressing plate shaft being connected with pressing plate body, its In, pressing plate shaft is be arranged in parallel with screw rod, and the left end of pressing plate shaft is rotated with box body by bearing one and is connected, and the right-hand member of pressing plate shaft passes through Bearing two is rotated with box body and is connected, and the right-hand member of pressing plate shaft stretches out box body and is connected with pressing plate rotation,
The full-automatic RNA libraries preparation facilities sequence measurement of second generation high-flux sequence is applied to, is comprised the following steps：
All reagents used in RNA library constructions are all placed in advance in the agent bin of box body bottom, RNA sample is through reverse transcription CDNA, DNA fragmentation, end repair, adjunction head, PCR amplification and after purification, the library for building is transferred to library by sampling probe In collecting pit, the closure at the top of box body is opened, the library for building is taken out by library transfer port, be then just put into sequencing It is sequenced on platform.
The described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is characterized in that：Text All reagents needed for prepared by storehouse are respectively placed in agent bin, and library collecting pit, waste liquid tank, the openend of agent bin are by masking foil Closing.
The described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is characterized in that：Text Storehouse collecting pit, sample cell, agent bin, the top of waste liquid tank set porous plate, and the left and right end of porous plate is fixed with inboard wall of cartridge respectively Set, the hole on porous plate is corresponding respectively with the openend of agent bin, waste liquid tank, sample cell, library collecting pit.
The described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is characterized in that：Box Internal wall is provided with the pressing plate limiting plate for limiting the pressing plate shaft anglec of rotation, and pressing plate limiting plate is located at pressing plate body lower section, and pressing plate is spacing The central shaft of plate is vertical with pressing plate shaft.
The described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is characterized in that：Bearing 3rd, the installation place of bearing four is respectively mounted sealing gasket.
The described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is characterized in that：Examination The reagent held respectively in the 1-9 of agent storehouse is that reagent set is unified or agent combination two, wherein,
Reagent set is unified：
Agent bin one：RNA Reverse Transcription mixed liquors,
Agent bin two：DNA fragmentation reagent mixed liquor；
Agent bin three：EDTA solution；
Agent bin four：Without DNase, RNase water；
Agent bin five：CMPμre Beads；
Agent bin six：Ethanol solution；
Agent bin seven：Repair and add A reagent mixed liquors in DNA ends；
Agent bin eight：Adaptor coupled reaction reagent mixed liquors；
Agent bin nine：PCR amplifing reagent mixed liquors,
Agent combination two is：
Agent bin one：RNA Reverse Transcription mixed liquors,
Agent bin two：DNA fragmentation reagent mixed liquor；
Agent bin three：EDTA solution；
Agent bin four：Nuclease-free Water；
Agent bin five：AMPμre Beads；
Agent bin six：Ethanol solution；
Agent bin seven：Repair and add A reagent mixed liquors in DNA ends；
Agent bin eight：Adaptor coupled reaction reagent mixed liquors；
Agent bin nine：PCR amplifing reagent mixed liquors.
Wherein,
Reagent set unification is preferably：
Reagent cabin one：The μ l of RNA Reverse Transcriptions mixed liquor 4；
Reagent cabin two：The ul of DNA fragmentation reagent mixed liquor 4.2, main component：10X dsDNA Fragmentase The μ l of Reaction B μ ffer 2, the μ l of BSA 0.2 of 100X 100 mg/ml, the μ l of dsDNA Fragmentase 2；
Reagent cabin three：The EDTA 10-20 μ l of 0.5M；
Reagent cabin four：Without DNase, RNase water 310-350 μ l；
Reagent cabin five：CMPμre Beads 310-350 μl；
Reagent cabin six：80% ethanol 900-1100 μ l；
Reagent cabin seven：Repair and add the μ l of A reagent mixed liquors 8.5, main component in DNA ends：10x End Repair Reaction Buffer 6.5 μl、End Prep Enzyme Mix 2 μl；
Reagent cabin eight：The μ l of Adaptor coupled reactions reagent mixed liquor 18.5, main component：T4 DNA ligase buffer 14 μl、T4 DNA ligase 2 μl、Adaptor 2.5 μl；
Reagent cabin nine：The μ l of PCR amplifing reagents mixed liquor 27, main component：HiFidelity 2X PCR Master Mix 25 μl、univesial primer 1μl、Index primer 1μl。
Agent combination two is preferably：
Reagent cabin one：The μ l of RNA Reverse Transcriptions mixed liquor 4；
Reagent cabin two：The μ l of DNA fragmentation reagent mixed liquor 4.2, it is 10X dsDNA Fragmentase Reaction B μ The μ l of ffer 2, the μ l of BSA 0.2 of 100X 100 mg/ml, the μ l of dsDNA Fragmentase 2；
Reagent cabin three：The EDTA 10-20 μ l of 0.5M；
Reagent cabin four： Nuclease-free Water 210-235 μl；
Reagent cabin five：AMPμre Beads 215-230 μl；
Reagent cabin six：80% ethanol 1120-1200 μ l；
Reagent cabin seven：DNA ends are repaired and add the ul of A reagent mixed liquors 18, and it is NEXTflex End-Repair ＆ Adenylation Buffer Mix 15 μl、NEXTflex™ End-Repair & Adenylation Enzyme Mix 3 μl；
Reagent cabin eight：The ul of Adaptor coupled reactions reagent mixed liquor 50, it is NEXTflex Ligase Enzyme Mix 47.5 μl、Adaptor 2.5 μl；
Reagent cabin nine：The ul of PCR amplifing reagents mixed liquor 14, its be the μ l of NEXTflex PCR Master Mix 12, NEXTflex™ Primer Mix 2 μl。
The described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is characterized in that：When From agent combination for the moment, sequence measurement specifically includes following steps：
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water, RNA concentration 150-300 ng/ μ l, RNA purity： OD260/OD280=1.78-2.0。
RNA libraries reagent preparation is held in the 1-9 of reagent cabin respectively.
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 10-15 μ l samples with pipettor is added to sample cell by injection port In, close injection port.
1st, RNA reverse transcriptions are cDNA
The RNA samples of 3.3-6.7 μ l in sample cell are transferred to reagent cabin by the small-sized pipettor of outside mechanical manipulation tray interior In one, while being shifted from reagent cabin four without in DNase, RNase water 9.3-12.7 μ l to reagent cabin one, it is ensured that cumulative volume is 20 μ l, react with the Reverse Transcription mixed liquor in reagent cabin one, and reaction condition is as follows：37℃ 15 min；85℃ 5 seconds；4 DEG C of holdings.
2nd, DNA fragmentationization reaction
The μ l of cDNA solution 15 in reagent cabin one are transferred to reagent cabin two by the small-sized pipettor of outside mechanical manipulation tray interior In, while in being shifted from reagent cabin four without the μ l of DNase, RNase water 0.8 to reagent cabin two, make reaction cumulative volume for 20 μ l, Reacted with the DNA fragmentation reagent mixed liquor in reagent cabin two.
Reaction condition：35-38 DEG C incubates 25-30 minutes.
Then the 0.5 M EDTA that 5 μ l are drawn from reagent cabin three add reagent cabin one, terminate enzymatic reaction.
3rd, cDNA fragment purifications
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid of the μ l of transferase 12 5 is put into reagent cabin two；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3）The magnetic force rack device of the outside machinery of control rises to the position of reagent cabin two, stands 5-10 minutes, makes magnetic bead and supernatant molten Liquid is separated；
4）The small-sized pipettor of tray interior draws the supernatant in reagent cabin two, is transferred to waste liquid tank；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin two, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in two, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without DNase, RNase water 35-40 μ l to reagent cabin one in, using box Body Internal Small-scale pipettor is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin two, magnetic bead is separated with the cDNA of wash-out.
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 25 in transfering reagent cabin two in reagent cabin seven, the then nothing in transfering reagent cabin four The μ l of DNase, RNase water 31.5 are inhaled and play 5-10 mixing, with examination up and down to reagent cabin seven using the small-sized pipettor of tray interior Repair reaction and add A reaction mixtures to be reacted in DNA ends in agent cabin seven；
2）Response procedures are as follows：12-15℃ 15-20 min；35-37℃ 15-20 min；70-72℃ 20-25 min；4℃ Keep.
5th, the purifying of end DNA plerosis
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 65 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin seven is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in seven, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 75 to reagent cabin seven in, using small-sized Pipettor is inhaled beat 5-10 times up and down, is stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin seven, magnetic bead is separated with the cDNA of wash-out.
6th, adaptor is connected
μ l of cDNA purification solutions 65 in reagent cabin seven are drawn in reagent cabin eight, is connected with the Adaptor in reagent cabin eight Reaction reagent mixed liquor reacts；
Inhaled up and down using the small-sized pipettor of tray interior and play 5-10 mixing；
Reaction condition：20-25 DEG C incubates 15-20 minutes.
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 83.5 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin eight is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin eight, 30-60 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in eight, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 30 to reagent cabin eight in, using box body Internal Small-scale pipettor is inhaled beat 5-10 times up and down, is stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into reagent cabin 8 positions, magnetic bead is separated with the cDNA of wash-out.
8th, PCR amplifications
The μ l of cDNA selective recoveries fragment 23 after the connection adaptor in reagent cabin eight are drawn, are transferred in reagent cabin nine, Reacted with the PCR amplifing reagents mixed liquor in reagent cabin nine.
Response procedures are as follows：95-98 DEG C of 30-45 s of predegeneration；95-98 DEG C of 10-15 s of denaturation；60-65 DEG C of annealing 30-40 s；Extend 70-72 DEG C of 30-40 s；6-10 circulation；Extend 70-72 DEG C of 4-5 min eventually.
9th, PCR primer purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid of the μ l of transferase 45 0 is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin nine is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin nine, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 5-10 minutes is stood, reagent cabin is drawn Supernatant in nine, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without DNase, RNase water 35-40 μ l to reagent cabin nine in, using box Body Internal Small-scale pipettor is inhaled beat 5-10 times up and down, is stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead is separated with PCR primer.
10th, PCR primer is taken out
PCR purification solution 25-30 μ l in reagent cabin nine are transferred to library collecting pit, will from outside by library transfer port PCR purification solutions remove box body, you can take carried out in microarray dataset fasciation into and sequencing, also RNA libraries can be stored in -20 DEG C preserve.
The described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is characterized in that：When During from agent combination two, sequence measurement specifically includes following steps：
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water, RNA concentration 150-300 ng/ μ l, RNA purity： OD260/OD280=1.78-2.0。
RNA libraries reagent preparation is held in the 1-9 of reagent cabin respectively.
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 10-15 μ l samples with pipettor is added to sample cell by injection port In, close injection port.
1st, RNA reverse transcriptions are cDNA
The RNA samples of 3.3-6.7 μ l in sample cell are transferred to reagent cabin by the small-sized pipettor of outside mechanical manipulation tray interior In one, while shifting Nuclease-free Water 9.3-12.7 μ l from reagent cabin four in reagent cabin one, it is ensured that overall Product is 20 μ l, is reacted with the Reverse Transcription mixed liquor in reagent cabin one, and reaction condition is as follows：37℃ 15 min；85 ℃ 5 seconds；4 DEG C of holdings.
2nd, DNA fragmentationization reaction
15 μ l cDNA solution in sample cell are transferred to reagent cabin two by the small-sized pipettor of outside mechanical manipulation tray interior In, while shifting the μ l of Nuclease-free Water 0.8 from reagent cabin four in reagent cabin two, making the reaction cumulative volume be 20 μ l, react with the DNA fragmentation reagent mixed liquor in reagent cabin two.
Reaction condition：35-38 DEG C incubates 30-40 minutes,.
Then the 0.5 M EDTA that 5 μ l are drawn from reagent cabin three add reagent cabin two, terminate enzymatic reaction.
3rd, DNA fragmentation purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 35 μ l is put into reagent cabin two；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3）The magnetic force rack device of the outside machinery of control rises to the position of reagent cabin two, stands 5-10 minutes, makes magnetic bead and supernatant molten Liquid is separated；
4）The small-sized pipettor of tray interior draws the supernatant in reagent cabin two, is transferred to waste liquid tank；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin two, 5-10 minutes is stood, magnetic bead and supernatant solution is separated.Draw reagent cabin Supernatant in two, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the Nuclease-free Water 30-35 μ l in absorption reagent cabin four to reagent cabin two, Inhaled up and down using the small-sized pipettor of tray interior and play 5-10 mixing, be stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin two, magnetic bead is separated with the cDNA of wash-out,
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 20 in transfering reagent cabin two in reagent cabin seven, using on the small-sized pipettor of tray interior 5-10 mixing is played in lower suction, with reagent cabin seven in DNA ends repair reaction and add A reaction mixtures reacted；
Response procedures are as follows：22-25℃ 18-22 min；70-72℃ 20-25 min；4 DEG C of holdings.
5th, the purifying of end DNA plerosis
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 80 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin seven is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in seven, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 60 in absorption reagent cabin four to reagent cabin seven, profit Inhaled up and down with small-sized pipettor and beaten 5-10 times, be stored at room temperature 5-10 minutes；
Magnetic frame is risen into the position of reagent cabin seven, magnetic bead is separated with the DNA of wash-out.
6th, adaptor is connected
1）μ l of cDNA purification solutions 50 in reagent cabin seven are drawn in reagent cabin eight, is connected with the Adaptor in reagent cabin eight Connect the reaction of reaction reagent mixed liquor；
2）Inhaled up and down using the small-sized pipettor of tray interior and play 10-15 mixing.
3）Reaction condition：22-25 DEG C incubates 15-20 minutes.
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 60 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin eight is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin eight, 30-60 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in eight, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 5-10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 30 in absorption reagent cabin four to reagent cabin eight, profit Inhaled up and down with the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into reagent cabin 8 positions, magnetic bead is separated with the cDNA of wash-out.
8th, PCR amplifications
The μ l of cDNA selective recoveries fragment 20 after the connection adaptor in reagent cabin eight are drawn, are transferred in reagent cabin nine, Reacted with the PCR amplifing reagents mixed liquor in reagent cabin nine.
Response procedures are as follows：95-98 DEG C of 2-2.5 min of predegeneration；95-98 DEG C of 30-40 s of denaturation；Annealing 60-65 ℃ 30-40 s；Extend 70-72 DEG C of 60-90 s；4-10 circulation；Extend 70-72 DEG C of 4-5 min eventually.
9th, PCR primer purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 40 μ l is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin nine is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin nine, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 5-10 minutes is stood, reagent cabin is drawn Supernatant in nine, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 5-10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the Nuclease-free Water 35-40 μ l in absorption reagent cabin four to reagent cabin nine, Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead is separated with PCR primer,
10th, PCR primer is taken out
PCR purification solution 25-30 μ l in reagent cabin nine are transferred to library collecting pit, shifting hole by library will from outside PCR purification solutions remove box body, you can take carried out in microarray dataset fasciation into and sequencing, also RNA libraries can be stored in -20 DEG C preserve.
The described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, it is characterized in that：Text Storehouse preparation method specifically also includes step：
Library Quality is detected：Fragment length distribution model in the RNA libraries agarose gel electrophoresis detection RNA libraries for preparing Enclose, if the Library Quality for building is good, can be directly sequenced.
Beneficial effect of the present invention：All reagents used in RNA library constructions are all placed in advance in box body bottom by the present invention In agent bin, RNA sample is cDNA, DNA fragmentation through reverse transcription, end is repaired, adjunction head, PCR are expanded and after purification, sampled Pin is transferred to the library for building in the collecting pit of library, and the closure at the top of box body is opened, and is taken out by library transfer port The library for building, then can just be put into carries out follow-up sequencing in microarray dataset.RNA sample is added into sample by injection port Seal injection port, waste liquid that building process is produced etc. behind pond to be all stored in the waste liquid tank of box body bottom, storehouse process of entirely founding a capital Completed in tray interior, whole process closing is not contacted with the external world, will not produce pollution, solved during scientific research and clinical test work Allow the pollution problem of people's rather headache, while liberated artificial, even one of ordinary skilled in the art can independently grasp Work is implemented, and improves efficiency.
（Four）Brief description of the drawings
The present invention is further illustrated below in conjunction with the accompanying drawings.
Accompanying drawing 1 is the structural representation of apparatus of the present invention；
Accompanying drawing 2 is the position relationship schematic diagram of pressing plate of the present invention and pressing plate limiting plate；
Accompanying drawing 3 is the position relationship schematic diagram of screw rod of the present invention and connector,
In figure, 1 box body, 11 injection ports, 2 pressing plate shafts, 21 bearings one, 23 bearings two, 24 pressing plate limiting plates, 25 pressing plate bodies, 26 Pressing plate shaft, 3 sampling probes, 31 pistons, 32 negative pressure generators, 33 second springs, 34 depression bar pins, 35 first springs, 36 pipelines, 4 spiral shells Bar, 41 bearings three, 42 bearings four, 44 connectors, 5 sample cells, 61 agent bins one, 62 agent bins two, 63 agent bins three, 64 examinations Agent storehouse four, 65 agent bins five, 66 agent bins six, 67 agent bins seven, 68 agent bins eight, 69 agent bins nine, 7 waste liquid tanks, 8 tinfoil paper, 9 porous plates, 12 library transfer ports, 13 library collecting pits.
（Five）Specific embodiment
The full-automatic RNA libraries preparation facilities that the present embodiment is applied to second generation high-flux sequence includes box body 1, the top of box body 1 Injection port 11 is set on face, it is characterised in that：Library transfer port 12 is also set up, being set in injection port 11, library transfer port 12 can Horizontally slip to close or open the closure of injection port 11, library transfer port 12, the bottom of box body 1 sets gradually 1 from left to right The sample cell 5,8 of individual library collecting pit 13,1 agent bin 61-69,1 waste liquid tank 7, agent bin 1, agent bin 2 62, Agent bin 3 63, agent bin 4 64, agent bin 5 65, agent bin 6 66, agent bin 7 67, agent bin 8 68, agent bin 9 69, sample cell 5 and library collecting pit 13, the top of waste liquid tank 7 are generally aligned in the same plane, and library prepares required all reagents It is placed in agent bin 61-69, to avoid reagent from being contaminated, library collecting pit 13, waste liquid tank 7, the openend of agent bin 61-69 Closed by tinfoil paper 8, the openend of sample cell 5 is unclosed.Sample cell 5 is oppositely arranged with injection port 11, it is possible to use pipettor will Storehouse sample is built to be added in sample cell 5 by injection port 11；Library collecting pit 13 is oppositely arranged with library transfer port 12, for containing The final library for building is put, can be suctioned out by the library that library transfer port 12 will be prepared with pipettor and be stored or directly use In sequencing；Waste liquid tank 7 is used to contain all waste liquids produced in the preparation process of library.
Agent bin 1, agent bin 2 62, agent bin 3 63, agent bin 4 64, agent bin 5 65, agent bin 6 66, the top of agent bin 7 67, agent bin 8 68, agent bin 9 69, waste liquid tank 7, sample cell 5 and library collecting pit 13 Porous plate 9 is provided with, porous plate 9 is fixedly connected with the inwall of box body 1, the hole on porous plate 9 corresponds to agent bin one respectively 61st, agent bin 2 62, agent bin 3 63, agent bin 4 64, agent bin 5 65, agent bin 6 66, agent bin 7 67, The openend of agent bin 8 68, agent bin 9 69, waste liquid tank 7, sample cell 5 and library collecting pit 13.It is respectively provided with box body 1 Screw rod 4, sampling probe 3, the left and right two ends of the screw rod 4 rotate with the corresponding side of box body 1 and are connected through bearing 3 41, bearing 4 42 respectively, Screw rod 4 is horizontally disposed with, and the right-hand member of screw rod 4 stretches out box body 1 and is connected with screw rod knob, and connector 44, the connector are set on screw rod 4 44 are connected with screw rod 4 by screw thread, rotate screw rod knob, connector 44 and the relative motion of screw rod 4, are set on the top surface of connector 44 Sampling probe accommodating hole 45, the central shaft of sampling probe accommodating hole 45 and the central axis of screw rod 4 are equipped with, sampling probe is housed The bottom of hole 45 is fixedly installed the first spring 35, and the first spring 35 is set with the axis of sampling probe accommodating hole 45, the first spring 35 are extended by the bottom up of sampling probe accommodating hole 35 and stretch out sampling probe accommodating hole 45, and sampling probe 3 is set in the first spring In 35, rotary screw knob, sampling probe is received with connector 44 in agent bin 61-69, sample cell 5, waste liquid tank 7, library Movement between collection pond 13, sampling probe 3 is connected by pipeline 36 with negative pressure generator 32, and negative pressure generator 32 includes Housing, piston 31 and piston rod, the free end that upper cover of piston rod has second spring 33, piston rod are fixedly connected depression bar pin 34 Left end, the right-hand member of depression bar pin 34 stretches out box body 1, for propelling piston bar, when depression bar pin 34 is not promoted, the second bullet Spring 33 pops into position depression bar pin 34, and the top of sampling probe 3 is provided with pressing plate 2, and pressing plate 2 includes pressing plate body 25 And the pressing plate shaft 26 being connected with pressing plate body 25, wherein, pressing plate shaft 26 be arranged in parallel with screw rod 4, pressing plate shaft 26 Left end is rotated with box body 1 by bearing 1 and is connected, and the right-hand member of pressing plate shaft 26 is by 1 turn of bearing 2 23 and box body Dynamic connection, the right-hand member of pressing plate shaft 26 stretches out box body 1 and is connected with pressing plate rotation.Pressing plate body 25 is used to press down on sampling Pin 3, rotary pressure plate knob, pressing plate shaft 26 presses down on sampling probe 3 with dynamic pressure plate body 25, not during rotary pressure plate knob, First spring 35 pops into position pressing plate body 25.Therefore the sampling probe 3 of tray interior, by piston 31, negative pressure generator 32nd, the effect of the spring 35 of second spring 33 and first realize between box body bottom difference agent bin 61-69 or so and on move down It is dynamic, complete the absorption and release of liquid.Agent bin 61-69, can be sequentially added into reverse transcription, DNA fragmentation, fragment ends and repair Various reagents needed for multiple, adjunction head, PCR amplifications and purifying, are then sealed with tinfoil paper 8, realize the full envelope of library preparation process Close, no pollution.Each agent bin is placed reagent and is followed successively by：Agent bin 1（Hold Reverse Transcription mixed liquor）, agent bin 2 62 （Hold DNA fragmentation reagent mixed liquor）, agent bin 3 63（Contain the EDTA of 0.5M）, agent bin 4 64（Contain without DNase, RNase water）, agent bin 5 65（Hold DNA fragmentation selective recovery magnetic bead）, agent bin 6 66（Contain 80% ethanol）, agent bin 7 67（DNA ends are held to repair and add A reagent mixed liquors）, agent bin 8 68（Hold the mixing of Adaptor coupled reactions reagent Liquid）, agent bin 9 69（Hold PCR amplifing reagent mixed liquors）.
The inwall of box body 1 is further fixedly arranged on the pressing plate limiting plate 24 for limiting the anglec of rotation of pressing plate shaft 26, pressing plate limit Position plate 24 is located at the lower section of pressing plate body 25, and the central shaft of pressing plate limiting plate 24 is vertical with pressing plate shaft 26, pressing plate shaft 26 When going to certain angle, pressing plate 25 is contacted with pressing plate limiting plate 24, and pressing plate shaft 26 can not continue rotation.
Full-automatic library preparation facilities of the invention is used cooperatively with outside mechanical part, and outside machinery is by control system control System, can in control system input instruction, set according to detecting step take liquid interval, the reagent that takes in which agent bin with And draw the volume of reagent etc..Sample injects sample cell 5 by injection port 11, closes injection port；What outside machinery basis set Sampling probe 3 inside step operation is between different agent bins or so and moves up and down, and completes the absorption and release of liquid, completes A series of flows prepared by library.The final library for preparing concentrates on library collecting pit 13, will be prepared using pipettor manually Good library suctions out, and stores or is directly sequenced.
Embodiment 1
The sequencing steps that the present embodiment is applied to the full-automatic RNA libraries preparation facilities of second generation high-flux sequence are as follows：
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water.Ng/ μ l, the RNA purity of RNA concentration 215：OD260/ OD280=1.85。
The reagent and volume held respectively in the 1-9 of reagent cabin be：
Reagent cabin one：The μ l of RNA Reverse Transcriptions mixed liquor 4（5×PrimeScript RT Master Mix (Perfect Real Time)；
Reagent cabin two：The ul of DNA fragmentation reagent mixed liquor 4.2（Main component：10X dsDNA Fragmentase Reaction Bμffer 2 μl、100X BSA (100 mg/ml) 0.2 μl、dsDNA Fragmentase 2 μl）；
Reagent cabin three：The μ l of EDTA 10 of 0.5M；
Reagent cabin four：Without the μ l of DNase, RNase water 310；
Reagent cabin five：CMPμre Beads 310μl；
Reagent cabin six：The μ l of 80% ethanol 900；
Reagent cabin seven：Repair and add the μ l of A reagent mixed liquors 8.5 in DNA ends（Main component：10x End Repair Reaction Buffer 6.5 μl、End Prep Enzyme Mix 2 μl）
Reagent cabin eight：The μ l of Adaptor coupled reactions reagent mixed liquor 18.5（Main component：T4 DNA ligase buffer 14 μl、T4 DNA ligase 2 μl、Adaptor 2.5 μl）；
Reagent cabin nine：The μ l of PCR amplifing reagents mixed liquor 27（Main component：HiFidelity 2X PCR Master Mix 25 μl、univesial primer 1μl、Index primer 1μl）.
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 10 μ l samples with pipettor is added in sample cell by injection port, Closing injection port.Box body is quickly got rid of under two with hand, to ensure that all reagents all concentrate on reagent bilge portion.（It is all anti-below Program is answered all to manipulate outside machinery by the software program of control system to realize.）
1st, RNA reverse transcriptions are cDNA
Be transferred to the RNA samples of 4.6 μ l in sample cell in reagent cabin one by the small-sized pipettor of outside mechanical manipulation tray interior, Shifted from reagent cabin four without in the μ l of DNase, RNase water 11.4 to reagent cabin one simultaneously, it is ensured that cumulative volume is 20 μ l, with examination Reverse Transcription mixed liquor in agent cabin one reacts, and reaction condition is as follows：37℃ 15 min；85℃ 5 seconds；4 DEG C keep.
2nd, DNA fragmentationization reaction
The μ l of cDNA solution 15 in reagent cabin one are transferred to reagent cabin two by the small-sized pipettor of outside mechanical manipulation tray interior In, while being shifted from reagent cabin four without in the μ l of DNase, RNase water 0.8 to reagent cabin two（Reaction cumulative volume is set to be 20 μ l）, reacted with the DNA fragmentation reagent mixed liquor in reagent cabin two.
Reaction condition：37 DEG C incubate 30 minutes.
Then the 0.5 M EDTA that 5 μ l are drawn from reagent cabin three add reagent cabin one, terminate enzymatic reaction.
3rd, DNA fragmentation purifying
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 10 times and thoroughly mix the CMPure magnetic beads in reagent cabin five, then The magnetic bead liquid of the μ l of transferase 12 5 is put into reagent cabin two；
2）The small-sized pipettor of tray interior is inhaled play 5 mixings up and down, is stored at room temperature 10 minutes；
3）The magnetic force rack device of the outside machinery of control rises to the position of reagent cabin two, stands 10 minutes, makes magnetic bead and supernatant solution Separate；
4）The small-sized pipettor of tray interior draws the supernatant in reagent cabin two, is transferred to waste liquid tank；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, 30 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin two, 5 minutes are stood, magnetic bead and supernatant solution is separated.Draw reagent cabin two In supernatant, be transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 35 to reagent cabin one in, using box body Internal Small-scale pipettor is inhaled play 5 mixings up and down, is stored at room temperature 10 minutes；
10）Magnetic frame is risen into the position of reagent cabin two, magnetic bead is separated with the cDNA of wash-out.
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 25 in transfering reagent cabin two in reagent cabin seven, the then nothing in transfering reagent cabin four The μ l of DNase, RNase water 31.5 are inhaled and play 5 mixings, with reagent cabin up and down to reagent cabin seven using the small-sized pipettor of tray interior Repair reaction and add A reaction mixtures to be reacted in DNA ends in seven；
2）Response procedures are as follows：12℃ 20 min；35℃ 20 min；70℃ 25 min；4 DEG C of holdings.
5th, the purifying of end DNA plerosis
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 10 times and thoroughly mix the CMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 65 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 5 mixings up and down, is stored at room temperature 10 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 5 minutes are stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin seven is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 30 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 5 minutes are stood, magnetic bead and supernatant solution is separated, reagent cabin seven is drawn In supernatant, be transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 75 to reagent cabin seven in, using small-sized Pipettor is inhaled make a call to 5 times up and down, is stored at room temperature 10 minutes；
10）Magnetic frame is risen into the position of reagent cabin seven, magnetic bead is separated with the cDNA of wash-out.
6th, adaptor is connected
1）μ l of cDNA purification solutions 65 in reagent cabin seven are drawn in reagent cabin eight, is connected with the Adaptor in reagent cabin eight Connect the reaction of reaction reagent mixed liquor；
2）Inhaled up and down using the small-sized pipettor of tray interior and play 5 mixings；
Reaction condition：20 DEG C incubate 20 minutes.
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 10 times and thoroughly mix the CMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 83.5 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and made a call to 5 times, be stored at room temperature 10 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 5 minutes are stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin eight is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin eight, 30 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 5 minutes are stood, magnetic bead and supernatant solution is separated, reagent cabin eight is drawn In supernatant, be transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 30 to reagent cabin eight in, using box body Internal Small-scale pipettor is inhaled make a call to 5 times up and down, is stored at room temperature 10 minutes；
10）Magnetic frame is risen into reagent cabin 8 positions, magnetic bead is separated with the cDNA of wash-out.
8th, PCR amplifications
The μ l of cDNA selective recoveries fragment 23 after the connection adaptor in reagent cabin eight are drawn, are transferred in reagent cabin nine, Reacted with the PCR amplifing reagents mixed liquor in reagent cabin nine.
Response procedures are as follows：95 DEG C of 45 s of predegeneration；95 DEG C of 15 s of denaturation；60 DEG C of 40 s of annealing；Extend 70 DEG C 40 s；6 circulations；Extend 70 DEG C of 5 min eventually.
9th, PCR primer purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid of the μ l of transferase 45 0 is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and made a call to 5 times, be stored at room temperature 10 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 5 minutes are stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin nine is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin nine, 30 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 5 minutes are stood, reagent cabin nine is drawn In supernatant, be transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 35 to reagent cabin nine in, using in box body The small-sized pipettor in portion is inhaled make a call to 5 times up and down, is stored at room temperature 10 minutes；
10）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead is separated with PCR primer.
10th, PCR primer is taken out
The μ l of PCR purification solutions 30 in reagent cabin nine are transferred to library collecting pit, it is from outside that PCR is pure by library transfer port Change solution remove box body, you can take carried out in microarray dataset fasciation into and sequencing, also cDNA library can be stored in -20 DEG C of guarantors Deposit.
11st, Library Quality detection
Fragment length distribution in the RNA libraries agarose gel electrophoresis detection RNA libraries for preparing, if the text for building Storehouse quality is good, can be directly sequenced.
Embodiment 2
The sequencing steps that the present embodiment is applied to the full-automatic RNA libraries preparation facilities of second generation high-flux sequence are as follows：
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water.Ng/ μ l, the RNA purity of RNA concentration 150：OD260/ OD280=2.0。
Reagent cabin three：The μ l of EDTA 20 of 0.5M；
Reagent cabin four：Without the μ l of DNase, RNase water 350；
Reagent cabin five：CMPμre Beads 350 μl；
Reagent cabin six：The μ l of 80% ethanol 1100；
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 15 μ l samples with pipettor is added in sample cell by injection port, Closing injection port.
1st, RNA reverse transcriptions are cDNA
The RNA samples of 6.7 μ l in sample cell are transferred to reagent cabin one by the small-sized pipettor of outside mechanical manipulation tray interior In, while being shifted from reagent cabin four without in the μ l of DNase, RNase water 9.3 to reagent cabin one, it is ensured that cumulative volume is 20 μ l, with Reverse Transcription mixed liquor in reagent cabin one reacts.
2nd, DNA fragmentationization reaction
Reaction condition：38 DEG C incubate 25 minutes.
3rd, DNA fragmentation purifying
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 15 times and thoroughly mix the CMPure magnetic beads in reagent cabin five, then The magnetic bead liquid of the μ l of transferase 12 5 is put into reagent cabin two；
2）The small-sized pipettor of tray interior is inhaled play 10 mixings up and down, is stored at room temperature 5 minutes；
3）The magnetic force rack device of the outside machinery of control rises to the position of reagent cabin two, stands 10 minutes, makes magnetic bead and supernatant solution Separate；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, 60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin two, 10 minutes are stood, magnetic bead and supernatant solution is separated.Draw reagent cabin two In supernatant, be transferred to waste liquid tank；
8）It is stored at room temperature 15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 40 to reagent cabin one in, using box body Internal Small-scale pipettor is inhaled play 10 mixings up and down, is stored at room temperature 5 minutes.
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 25 in transfering reagent cabin two in reagent cabin seven, the then nothing in transfering reagent cabin four The μ l of DNase, RNase water 31.5 are inhaled and play 10 mixings, with reagent up and down to reagent cabin seven using the small-sized pipettor of tray interior Repair reaction and add A reaction mixtures to be reacted in DNA ends in cabin seven；
2）Response procedures are as follows： 15℃ 15 min； 37℃ 15 min； 72℃ 20 min；4 DEG C of holdings.
5th, the purifying of end DNA plerosis
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 15 times and thoroughly mix the CMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 65 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 10 mixings up and down, is stored at room temperature 5 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 10 minutes are stood, magnetic bead and supernatant solution is separated；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 10 minutes are stood, magnetic bead and supernatant solution is separated, reagent cabin seven is drawn In supernatant, be transferred to waste liquid tank；
8）It is stored at room temperature 15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 75 to reagent cabin seven in, using small-sized Pipettor is inhaled make a call to 10 times up and down, is stored at room temperature 5 minutes.
6th, adaptor is connected
Inhaled up and down using the small-sized pipettor of tray interior and play 10 mixings；
Reaction condition：25 DEG C incubate 15 minutes.
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 15 times and thoroughly mix the CMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 83.5 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and made a call to 10 times, be stored at room temperature 5 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 10 minutes are stood, magnetic bead and supernatant solution is separated；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin eight, 60 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 10 minutes are stood, magnetic bead and supernatant solution is separated, reagent cabin eight is drawn In supernatant, be transferred to waste liquid tank；
8）It is stored at room temperature 15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 30 to reagent cabin eight in, using box body Internal Small-scale pipettor is inhaled make a call to 10 times up and down, is stored at room temperature 5 minutes.
8th, PCR amplifications
Response procedures are as follows：98 DEG C of 30 s of predegeneration；98 DEG C of 10s of denaturation；65 DEG C of 30s of annealing；Extend 72 DEG C of 30 s； 10 circulations；Extend 72 DEG C of 4 min eventually.
9th, PCR primer purifying
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 15 times and thoroughly mix the CMPure magnetic beads in reagent cabin five, then The magnetic bead liquid of the μ l of transferase 45 0 is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and made a call to 10 times, be stored at room temperature 5 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 10 minutes are stood, magnetic bead and supernatant solution is separated；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin nine, 60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 10 minutes are stood, reagent cabin nine is drawn In supernatant, be transferred to waste liquid tank；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 40 to reagent cabin nine in, using box body Internal Small-scale pipettor is inhaled make a call to 10 times up and down, is stored at room temperature 5 minutes.
10th, PCR primer is taken out
The μ l of PCR purification solutions 30 in reagent cabin nine are transferred to library collecting pit, by library transfer port from outside by PCR Purification solution remove box body, you can take carried out in microarray dataset fasciation into and sequencing, also cDNA library can be stored in -20 DEG C Preserve.
Other are same as Example 1.
Embodiment 3
The sequencing steps that the present embodiment is applied to the full-automatic RNA libraries preparation facilities of second generation high-flux sequence are as follows：
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water.Ng/ μ l, the RNA purity of RNA concentration 300：OD260/ OD280=1.78。
Reagent cabin three：The μ l of EDTA 15 of 0.5M；
Reagent cabin four：Without the μ l of DNase, RNase water 330；
Reagent cabin five：CMPμre Beads 330 μl；
Reagent cabin six：The μ l of 80% ethanol 1000；
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 13 μ l samples with pipettor is added in sample cell by injection port, Closing injection port.
1st, RNA reverse transcriptions are cDNA
Be transferred to the RNA samples of 3.3 μ l in sample cell in reagent cabin one by the small-sized pipettor of outside mechanical manipulation tray interior, Shifted from reagent cabin four without in the μ l of DNase, RNase water 12.7 to reagent cabin one simultaneously, it is ensured that cumulative volume is 20 μ l, with examination Reverse Transcription mixed liquor in agent cabin one reacts.
3rd, DNA fragmentation purifying
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 38 to reagent cabin one in, using in box body The small-sized pipettor in portion is inhaled play 8 mixings up and down, is stored at room temperature 7 minutes.
9th, PCR primer purifying
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 35 to reagent cabin nine in, using box body Internal Small-scale pipettor is inhaled make a call to 8 times up and down, is stored at room temperature 8 minutes.
10th, PCR primer is taken out
The μ l of PCR purification solutions 28 in reagent cabin nine are transferred to library collecting pit, by library transfer port from outside by PCR Purification solution remove box body, you can take carried out in microarray dataset fasciation into and sequencing, also cDNA library can be stored in -20 DEG C Preserve.
Other are same as Example 1.
Embodiment 4
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water.Ng/ μ l, the RNA purity of RNA concentration 150：OD260/ OD280=2.0。
The reagent and volume held respectively in the 1-9 of reagent cabin be：
Reagent cabin one：The μ l of RNA Reverse Transcriptions mixed liquor 4（5×PrimeScript RT Master Mix (Perfect Real Time)；
Reagent cabin two：The μ l of DNA fragmentation reagent mixed liquor 4.2（10X dsDNA Fragmentase Reaction Bμffer 2 μl、100X BSA (100 mg/ml) 0.2 μl、dsDNA Fragmentase 2 μl）；
Reagent cabin three：The μ l of EDTA 10 of 0.5M；
Reagent cabin four： Nuclease-free Water 210μl；
Reagent cabin five：AMPμre Beads 215μl；
Reagent cabin six：The μ l of 80% ethanol 1120；
Reagent cabin seven：Repair and add the ul of A reagent mixed liquors 18 in DNA ends（NEXTflex™ End-Repair & Adenylation Buffer Mix 15 μl、NEXTflex™ End-Repair & Adenylation Enzyme Mix 3 μl）；
Reagent cabin eight：The ul of Adaptor coupled reactions reagent mixed liquor 50（NEXTflex™ Ligase Enzyme Mix 47.5 μl、Adaptor 2.5 μl）；
Reagent cabin nine：The ul of PCR amplifing reagents mixed liquor 14（NEXTflex™ PCR Master Mix 12μl、NEXTflex™ Primer Mix 2 μl）.
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 10 μ l samples with pipettor is added in sample cell by injection port, Closing injection port.Box body is quickly got rid of under two with hand, to ensure that all reagents all concentrate on reagent bilge portion.（It is all anti-below Program is answered all to manipulate outside machinery by the software program of control system to realize.）
1st, RNA reverse transcriptions are cDNA
The RNA samples of 6.7 μ l in sample cell are transferred to reagent cabin one by the small-sized pipettor of outside mechanical manipulation tray interior In, while shifting the μ l of Nuclease-free Water 9.3 from reagent cabin four in reagent cabin one, it is ensured that cumulative volume is 20 Reverse Transcription mixed liquor in μ l, with reagent cabin one reacts, and reaction condition is as follows：37℃ 15 min；85℃ 5 seconds；4 DEG C of holdings.
2nd, DNA fragmentationization reaction
15 μ l cDNA solution in sample cell are transferred to reagent cabin two by the small-sized pipettor of outside mechanical manipulation tray interior In, while shifting the μ l of Nuclease-free Water 0.8 from reagent cabin four in reagent cabin two（The reaction cumulative volume is set to be 20 μl）, reacted with the DNA fragmentation reagent mixed liquor in reagent cabin two.
Reaction condition：35 DEG C incubate 40 minutes.
Then the 0.5 M EDTA that 5 μ l are drawn from reagent cabin three add reagent cabin two, terminate enzymatic reaction.
3rd, DNA fragmentation purifying
1) inhaled up and down by the small-sized pipettor of tray interior and make a call to 10 times and thoroughly mix the AMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 35 μ l is put into reagent cabin two；
2) the small-sized pipettor of tray interior is inhaled play 5 mixings up and down, is stored at room temperature 10 minutes；
3) control the magnetic force rack device of outside machinery to rise to the position of reagent cabin two, stand 5 minutes, make magnetic bead and supernatant solution point From；
4) the small-sized pipettor of tray interior draws the supernatant in reagent cabin two, is transferred to waste liquid tank；
5) magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, stands 30 seconds；
6) magnetic frame is risen into the position of reagent cabin two, stands 5 minutes, separate magnetic bead and supernatant solution.Draw reagent cabin two In supernatant, be transferred to waste liquid tank；
7) repeat step 5,6；
8) it is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9) magnetic frame is reduced, in the μ l of Nuclease-free Water 30 in absorption reagent cabin four to reagent cabin two, profit Inhaled up and down with the small-sized pipettor of tray interior and play 5 mixings, be stored at room temperature 10 minutes；
10) magnetic frame is risen into the position of reagent cabin two, magnetic bead is separated with the cDNA of wash-out.
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 20 in transfering reagent cabin two in reagent cabin seven, using on the small-sized pipettor of tray interior 5 mixings are played in lower suction, with reagent cabin seven in DNA ends repair reaction and add A reaction mixtures reacted；
2) response procedures are as follows：22℃ 22 min；70℃ 25 min；4 DEG C of holdings.
5th, the purifying of end DNA plerosis
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 10 times and thoroughly mix the AMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 80 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 5 mixings up and down, is stored at room temperature 10 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 5 minutes are stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin seven is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 30 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 5 minutes are stood, magnetic bead and supernatant solution is separated, reagent cabin seven is drawn In supernatant, be transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 60 in absorption reagent cabin four to reagent cabin seven, profit Inhaled up and down with small-sized pipettor and made a call to 5 times, be stored at room temperature 10 minutes；
10) magnetic frame is risen into the position of reagent cabin seven, magnetic bead is separated with the DNA of wash-out.
6th, adaptor is connected
1) μ l of cDNA purification solutions 50 in reagent cabin seven are drawn in reagent cabin eight, is connected with the Adaptor in reagent cabin eight Connect the reaction of reaction reagent mixed liquor；
2) inhaled up and down using the small-sized pipettor of tray interior and play 10 mixings；
3) reaction condition：22 DEG C incubate 20 minutes.
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 10 times and thoroughly mix the AMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 60 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and made a call to 5 times, be stored at room temperature 10 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 5 minutes are stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin eight is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin eight, 30 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 5 minutes are stood, magnetic bead and supernatant solution is separated, reagent cabin eight is drawn In supernatant, be transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 5 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 30 in absorption reagent cabin four to reagent cabin eight, profit Inhaled up and down with the small-sized pipettor of tray interior and made a call to 5 times, be stored at room temperature 10 minutes；
10）Magnetic frame is risen into reagent cabin 8 positions, magnetic bead is separated with the cDNA of wash-out.
8th, PCR amplifications
The μ l of cDNA selective recoveries fragment 20 after the connection adaptor in reagent cabin eight are drawn, are transferred in reagent cabin nine, Reacted with the PCR amplifing reagents mixed liquor in reagent cabin nine.
Response procedures are as follows：95 DEG C of 2.5 min of predegeneration；95 DEG C of 40 s of denaturation；60 DEG C of 40 s of annealing；Extend 70℃ 90 s；4 circulations；Extend 70 DEG C of 5 min eventually.
9th, PCR primer purifying
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 10 times and thoroughly mix the AMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 40 μ l is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and made a call to 5 times, be stored at room temperature 10 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 5 minutes are stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin nine is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin nine, 30 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 5 minutes are stood, reagent cabin nine is drawn In supernatant, be transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 5 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 35 in absorption reagent cabin four to reagent cabin nine, is utilized The small-sized pipettor of tray interior is inhaled make a call to 5 times up and down, is stored at room temperature 10 minutes；
10）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead is separated with PCR primer.
10th, PCR primer is taken out
The μ l of PCR purification solutions 25 in reagent cabin nine are transferred to library collecting pit, it is pure by PCR from outside to shift hole by library Change solution remove box body, you can take carried out in microarray dataset fasciation into and sequencing, also -20 DEG C of guarantors can be stored in RNA libraries Deposit.
11st, Library Quality detection
Fragment length distribution in the RNA libraries agarose gel electrophoresis detection RNA libraries for preparing, if the text for building Storehouse quality is good, can be directly sequenced.
Embodiment 5
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water.Ng/ μ l, the RNA purity of RNA concentration 300：OD260/ OD280=1.78。
The reagent and volume held respectively in the 1-9 of reagent cabin be：
Reagent cabin one：The μ l of RNA Reverse Transcriptions mixed liquor 4（5×PrimeScript RT Master Mix (Perfect Real Time)；
Reagent cabin two：The μ l of DNA fragmentation reagent mixed liquor 4.2（10X dsDNA Fragmentase Reaction Bμffer 2 μl、100X BSA (100 mg/ml) 0.2 μl、dsDNA Fragmentase 2 μl）；
Reagent cabin three：The EDTA 10-20 μ l of 0.5M；
Reagent cabin four： Nuclease-free Water 210-235 μl；
Reagent cabin five：AMPμre Beads 215-230 μl；
Reagent cabin six：80% ethanol 1120-1200 μ l；
Reagent cabin seven：Repair and add the ul of A reagent mixed liquors 18 in DNA ends（NEXTflex™ End-Repair & Adenylation Buffer Mix 15 μl、NEXTflex™ End-Repair & Adenylation Enzyme Mix 3 μl）；
Reagent cabin eight：The ul of Adaptor coupled reactions reagent mixed liquor 50（NEXTflex™ Ligase Enzyme Mix 47.5 μl、Adaptor 2.5 μl）；
Reagent cabin nine：The ul of PCR amplifing reagents mixed liquor 14（NEXTflex™ PCR Master Mix 12μl、NEXTflex™ Primer Mix 2 μl）.
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 10-15 μ l samples with pipettor is added to sample cell by injection port In, close injection port.Box body is quickly got rid of under two with hand, to ensure that all reagents all concentrate on reagent bilge portion.（Hereinafter own Response procedures all manipulate outside machinery to realize by the software program of control system.）
1st, RNA reverse transcriptions are cDNA
The RNA samples of 3.3-6.7 μ l in sample cell are transferred to reagent cabin by the small-sized pipettor of outside mechanical manipulation tray interior In one, while shifting Nuclease-free Water 9.3-12.7 μ l from reagent cabin four in reagent cabin one, it is ensured that overall Product is 20 μ l, is reacted with the Reverse Transcription mixed liquor in reagent cabin one, and reaction condition is as follows：37℃ 15 min；85 ℃ 5 seconds；4 DEG C of holdings.
2nd, DNA fragmentationization reaction
15 μ l cDNA solution in sample cell are transferred to reagent cabin two by the small-sized pipettor of outside mechanical manipulation tray interior In, while shifting the μ l of Nuclease-free Water 0.8 from reagent cabin four in reagent cabin two（The reaction cumulative volume is set to be 20 μl）, reacted with the DNA fragmentation reagent mixed liquor in reagent cabin two.
Reaction condition：35-38 DEG C incubates 30-40 minutes.
Then the 0.5 M EDTA that 5 μ l are drawn from reagent cabin three add reagent cabin two, terminate enzymatic reaction.
3rd, DNA fragmentation purifying
1) inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 35 μ l is put into reagent cabin two；
2) the small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3) control the magnetic force rack device of outside machinery to rise to the position of reagent cabin two, stand 5-10 minutes, make magnetic bead and supernatant molten Liquid is separated；
4) the small-sized pipettor of tray interior draws the supernatant in reagent cabin two, is transferred to waste liquid tank；
5) magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, stands 30-60 seconds；
6) magnetic frame is risen into the position of reagent cabin two, stands 5-10 minutes, separate magnetic bead and supernatant solution.Draw reagent cabin Supernatant in two, is transferred to waste liquid tank；
7) repeat step 5,6；
8) it is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9) magnetic frame is reduced, in the Nuclease-free Water 30-35 μ l in absorption reagent cabin four to reagent cabin two, Inhaled up and down using the small-sized pipettor of tray interior and play 5-10 mixing, be stored at room temperature 5-10 minutes；
10) magnetic frame is risen into the position of reagent cabin two, magnetic bead is separated with PCR primer.
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 20 in transfering reagent cabin two in reagent cabin seven, using on the small-sized pipettor of tray interior 5-10 mixing is played in lower suction, with reagent cabin seven in DNA ends repair reaction and add A reaction mixtures reacted；
2) response procedures are as follows：22-25℃ 18-22 min；70-72℃ 20-25 min；4 DEG C of holdings.
5th, the purifying of end DNA plerosis
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 80 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin seven is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in seven, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 60 in absorption reagent cabin four to reagent cabin seven, profit Inhaled up and down with small-sized pipettor and beaten 5-10 times, be stored at room temperature 5-10 minutes；
10) magnetic frame is risen into the position of reagent cabin seven, magnetic bead is separated with the DNA of wash-out.
6th, adaptor is connected
1) μ l of cDNA purification solutions 50 in reagent cabin seven are drawn in reagent cabin eight, is connected with the Adaptor in reagent cabin eight Connect the reaction of reaction reagent mixed liquor；
2) inhaled up and down using the small-sized pipettor of tray interior and play 10-15 mixing；
3) reaction condition：22-25 DEG C incubates 15-20 minutes.
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 60 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin eight is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin eight, 30-60 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in eight, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 5-10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 30 in absorption reagent cabin four to reagent cabin eight, profit Inhaled up and down with the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into reagent cabin 8 positions, magnetic bead is separated with PCR primer.
8th, PCR amplifications
The μ l of cDNA selective recoveries fragment 20 after the connection adaptor in reagent cabin eight are drawn, are transferred in reagent cabin nine, Reacted with the PCR amplifing reagents mixed liquor in reagent cabin nine.
Response procedures are as follows：95-98 DEG C of 2-2.5 min of predegeneration；95-98 DEG C of 30-40 s of denaturation；Annealing 60-65 ℃ 30-40 s；Extend 70-72 DEG C of 60-90 s；4-10 circulation；Extend 70-72 DEG C of 4-5 min eventually.
9th, PCR primer purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 40 μ l is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin nine is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin nine, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 5-10 minutes is stood, reagent cabin is drawn Supernatant in nine, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 5-10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the Nuclease-free Water 35-40 μ l in absorption reagent cabin four to reagent cabin nine, Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead is separated with PCR primer.
10th, PCR primer is taken out
PCR purification solution 25-30 μ l in reagent cabin nine are transferred to library collecting pit, shifting hole by library will from outside PCR purification solutions remove box body, you can take carried out in microarray dataset fasciation into and sequencing, cDNA library can be also stored in- 20 DEG C of preservations.
11st, Library Quality detection
The cDNA library for preparing detects the fragment length distribution in cDNA library with agarose gel electrophoresis, if build Library Quality is good, can be directly sequenced.
Embodiment 5
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water.Ng/ μ l, the RNA purity of RNA concentration 300：OD260/ OD280=1.78。
Reagent cabin three：The μ l of EDTA 20 of 0.5M；
Reagent cabin four： Nuclease-free Water 235 μl；
Reagent cabin five：AMPμre Beads 230 μl；
Reagent cabin six：The μ l of 80% ethanol 1200.
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 15 μ l samples with pipettor is added in sample cell by injection port, Closing injection port.
1st, RNA reverse transcriptions are cDNA
The RNA samples of 6.6 μ l in sample cell are transferred to reagent cabin one by the small-sized pipettor of outside mechanical manipulation tray interior In, while shifting the μ l of Nuclease-free Water 9.4 from reagent cabin four in reagent cabin one, it is ensured that cumulative volume is 20 μl。
2nd, DNA fragmentationization reaction
Reaction condition：38 DEG C incubate 30 minutes.
3rd, DNA fragmentation purifying
1) inhaled up and down by the small-sized pipettor of tray interior and make a call to 15 times and thoroughly mix the AMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 35 μ l is put into reagent cabin two；
2) the small-sized pipettor of tray interior is inhaled play 10 mixings up and down, is stored at room temperature 5 minutes；
3) control the magnetic force rack device of outside machinery to rise to the position of reagent cabin two, stand 10 minutes, make magnetic bead and supernatant solution Separate；
5) magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, stands 60 seconds；
6) magnetic frame is risen into the position of reagent cabin two, stands 10 minutes, separate magnetic bead and supernatant solution.Draw reagent cabin two In supernatant, be transferred to waste liquid tank；
8) it is stored at room temperature 15 minutes, magnetic bead is dried in atmosphere；
9) magnetic frame is reduced, in the μ l of Nuclease-free Water 35 in absorption reagent cabin four to reagent cabin two, profit Inhaled up and down with the small-sized pipettor of tray interior and play 10 mixings, be stored at room temperature 5 minutes.
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 20 in transfering reagent cabin two in reagent cabin seven, using on the small-sized pipettor of tray interior 10 mixings are played in lower suction, with reagent cabin seven in DNA ends repair reaction and add A reaction mixtures reacted；
2) response procedures are as follows： 25℃ 18 min； 72℃ 20 min；4 DEG C of holdings.
5th, the purifying of end DNA plerosis
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 15 times and thoroughly mix the AMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 80 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 10 mixings up and down, is stored at room temperature 5 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 10 minutes are stood, magnetic bead and supernatant solution is separated；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 10 minutes are stood, magnetic bead and supernatant solution is separated, reagent cabin seven is drawn In supernatant, be transferred to waste liquid tank；
8）It is stored at room temperature 15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 60 in absorption reagent cabin four to reagent cabin seven, profit Inhaled up and down with small-sized pipettor and made a call to 10 times, be stored at room temperature 5 minutes.
6th, adaptor is connected
2) inhaled up and down using the small-sized pipettor of tray interior and play 15 mixings；
3) reaction condition：25 DEG C incubate 15 minutes.
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 15 times and thoroughly mix the AMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 60 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and made a call to 10 times, be stored at room temperature 5 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 10 minutes are stood, magnetic bead and supernatant solution is separated；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin eight, 60 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 10 minutes are stood, magnetic bead and supernatant solution is separated, reagent cabin eight is drawn In supernatant, be transferred to waste liquid tank；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 30 in absorption reagent cabin four to reagent cabin eight, profit Inhaled up and down with the small-sized pipettor of tray interior and made a call to 10 times, be stored at room temperature 5 minutes.
8th, PCR amplifications
Response procedures are as follows：98 DEG C of 2 min of predegeneration；98 DEG C of 30 s of denaturation；65 DEG C of 30s of annealing；Extend 72 DEG C 60 s；10 circulations；Extend 72 DEG C of 4 min eventually.
9th, PCR primer purifying
1）Inhaled up and down by the small-sized pipettor of tray interior and make a call to 15 times and thoroughly mix the AMPure magnetic beads in reagent cabin five, then The magnetic bead liquid for shifting 40 μ l is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and made a call to 10 times, be stored at room temperature 5 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 10 minutes are stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin nine is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin nine, 60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 10 minutes are stood, reagent cabin nine is drawn In supernatant, be transferred to waste liquid tank；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 40 in absorption reagent cabin four to reagent cabin nine, is utilized The small-sized pipettor of tray interior is inhaled make a call to 10 times up and down, is stored at room temperature 5 minutes；
10）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead is separated with PCR primer.
10th, PCR primer is taken out
The μ l of PCR purification solutions 30 in reagent cabin nine are transferred to library collecting pit, hole is shifted from outside by PCR by library Purifying cDNA solution removes box body, you can take carried out in microarray dataset fasciation into and sequencing, cDNA library can be also stored in- 20 DEG C of preservations.
Other are same as Example 4.

Claims (10)

1. a kind of full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence, including box body, on the top surface of box body Injection port is set, it is characterised in that：Library transfer port is also set up, being set in injection port, library transfer port can horizontally slip to seal Close or open injection port, the closure of library transfer port, box body bottom be respectively provided with 1 library collecting pit, 1 sample cell, 9 Agent bin, 1 waste liquid tank, wherein, sample cell is oppositely arranged with injection port, and library collecting pit is oppositely arranged with library transfer port, Screw rod, sampling probe are respectively provided with box body, the left and right two ends of screw rod turn through bearing three, bearing four and box body corresponding side respectively Dynamic connection, screw rod is horizontally disposed with, and the right-hand member of screw rod stretches out box body and is connected with screw rod knob, connector is set on screw rod, the connection Part is connected with screw rod by screw thread, is provided with sampling probe accommodating hole on the top surface of connector, the central shaft of sampling probe accommodating hole with The central axis of screw rod, sampling probe accommodating hole bottom is fixedly installed the first spring, the first spring and sampling probe accommodating hole axis Set, the first spring is extended by sampling probe accommodating hole bottom up and stretches out sampling probe accommodating hole, and sampling probe is set in the first bullet In spring, sampling probe is connected by pipeline with negative pressure generator, and negative pressure generator includes housing, piston and piston rod, on piston rod Second spring is cased with, the free end of piston rod is fixedly connected the left end of depression bar pin, and the right-hand member of depression bar pin stretches out box body, for advancing Piston rod, is provided with pressing plate above sampling probe, pressing plate includes pressing plate body and the pressing plate shaft being connected with pressing plate body, wherein, Pressing plate shaft is be arranged in parallel with screw rod, and the left end of pressing plate shaft is rotated with box body by bearing one and is connected, and the right-hand member of pressing plate shaft passes through axle Hold two to be connected with box body rotation, the right-hand member of pressing plate shaft stretches out box body and is connected with pressing plate rotation,
The full-automatic RNA libraries preparation facilities sequence measurement of second generation high-flux sequence is applied to, is comprised the following steps：
All reagents used in RNA library constructions are all placed in advance in the agent bin of box body bottom, RNA sample is through reverse transcription CDNA, DNA fragmentation, end repair, adjunction head, PCR amplification and after purification, the library for building is transferred to library by sampling probe In collecting pit, the closure at the top of box body is opened, the library for building is taken out by library transfer port, be then just put into sequencing It is sequenced on platform.
2. the full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence according to claim 1, its feature It is：All reagents needed for prepared by library are respectively placed in agent bin, library collecting pit, waste liquid tank, the openend of agent bin Closed by masking foil.
3. the full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence according to claim 2, its feature It is：Library collecting pit, sample cell, agent bin, waste liquid tank top set porous plate, the left and right end of porous plate respectively with box body Inwall is fixedly installed, and the hole on porous plate is corresponding respectively with the openend of agent bin, waste liquid tank, sample cell, library collecting pit.
4. the full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence according to claim 3, its feature It is：Inboard wall of cartridge is provided with the pressing plate limiting plate for limiting the pressing plate shaft anglec of rotation, and pressing plate limiting plate is located at pressing plate body lower section, pressure The central shaft of plate limiting plate is vertical with pressing plate shaft.
5. the full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence according to claim 4, its feature It is：Bearing three, the installation place of bearing four are respectively mounted sealing gasket.
6. according to any described full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence of claim 1-5, It is characterized in that：The reagent held respectively in agent bin 1-9 is that reagent set is unified or agent combination two, wherein,
Reagent set is unified：
Agent bin one：RNA Reverse Transcription mixed liquors；
Agent bin two：DNA fragmentation reagent mixed liquor；
Agent bin three：EDTA solution；
Agent bin four：Without DNase, RNase water；
Agent bin five：CMPμre Beads；
Agent bin six：Ethanol solution；
Agent bin seven：Repair and add A reagent mixed liquors in DNA ends；
Agent bin eight：Adaptor coupled reaction reagent mixed liquors；
Agent bin nine：PCR amplifing reagent mixed liquors,
Agent combination two is：
Agent bin one：RNA Reverse Transcription mixed liquors,
Agent bin two：DNA fragmentation reagent mixed liquor；
Agent bin three：EDTA solution；
Agent bin four：Nuclease-free Water；
Agent bin five：AMPμre Beads；
Agent bin six：Ethanol solution；
Agent bin seven：Repair and add A reagent mixed liquors in DNA ends；
Agent bin eight：Adaptor coupled reaction reagent mixed liquors；
Agent bin nine：PCR amplifing reagent mixed liquors.
7. the full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence according to claim 6, its feature It is：The reagent held respectively in agent bin 1-9 is that reagent set is unified or agent combination two, wherein,
Reagent set is unified：
Reagent cabin one：The μ l of RNA Reverse Transcriptions mixed liquor 4；
Reagent cabin two：The ul of DNA fragmentation reagent mixed liquor 4.2, main component：10X dsDNA Fragmentase The μ l of Reaction B μ ffer 2, the μ l of BSA 0.2 of 100X 100 mg/ml, the μ l of dsDNA Fragmentase 2；
Reagent cabin three：The EDTA 10-20 μ l of 0.5M；
Reagent cabin four：Without DNase, RNase water 310-350 μ l；
Reagent cabin five：CMPμre Beads 310-350 μl；
Reagent cabin six：80% ethanol 900-1100 μ l；
Reagent cabin seven：Repair and add the μ l of A reagent mixed liquors 8.5, main component in DNA ends：10x End Repair The μ l of Reaction Buffer 6.5, the μ l of End Prep Enzyme Mix 2,
Reagent cabin eight：The μ l of Adaptor coupled reactions reagent mixed liquor 18.5, main component：T4 DNA ligase buffer 14 μl、T4 DNA ligase 2 μl、Adaptor 2.5 μl；
Reagent cabin nine：The μ l of PCR amplifing reagents mixed liquor 27, main component：HiFidelity 2X PCR Master Mix 25 μ l, the μ l of univesial primer 1, the μ l of Index primer 1,
Agent combination two is：
Reagent cabin one：The μ l of RNA Reverse Transcriptions mixed liquor 4；
Reagent cabin two：The μ l of DNA fragmentation reagent mixed liquor 4.2, it is 10X dsDNA Fragmentase Reaction B μ The μ l of ffer 2, the μ l of BSA 0.2 of 100X 100 mg/ml, the μ l of dsDNA Fragmentase 2；
Reagent cabin three：The EDTA 10-20 μ l of 0.5M；
Reagent cabin four： Nuclease-free Water 210-235 μl；
Reagent cabin five：AMPμre Beads 215-230 μl；
Reagent cabin six：80% ethanol 1120-1200 μ l；
Reagent cabin seven：DNA ends are repaired and add the ul of A reagent mixed liquors 18, and it is NEXTflex End-Repair ＆ Adenylation Buffer Mix 15 μl、NEXTflex™ End-Repair & Adenylation Enzyme Mix 3 μl；
Reagent cabin eight：The ul of Adaptor coupled reactions reagent mixed liquor 50, it is NEXTflex Ligase Enzyme Mix 47.5 μl、Adaptor 2.5 μl；
Reagent cabin nine：The ul of PCR amplifing reagents mixed liquor 14, its be the μ l of NEXTflex PCR Master Mix 12, NEXTflex™ Primer Mix 2 μl。
8. the full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence according to claim 6, its feature It is：When from agent combination, for the moment, sequence measurement specifically includes following steps：
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water, RNA concentration 150-300 ng/ μ l, RNA purity： OD260/OD280=1.78-2.0,
Reagent is held respectively in the 1-9 of reagent cabin,
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 10-15 μ l samples with pipettor is added to sample cell by injection port In, injection port is closed,
1st, RNA reverse transcriptions are cDNA
The RNA samples of 3.3-6.7 μ l in sample cell are transferred to reagent cabin by the small-sized pipettor of outside mechanical manipulation tray interior In one, while being shifted from reagent cabin four without in DNase, RNase water 9.3-12.7 μ l to reagent cabin one, it is ensured that cumulative volume is 20 μ l, react with the Reverse Transcription mixed liquor in reagent cabin one, and reaction condition is as follows：37℃ 15 min；85℃ 5 seconds；4 DEG C of holdings,
2nd, DNA fragmentationization reaction
The μ l of cDNA solution 15 in reagent cabin one are transferred to reagent cabin two by the small-sized pipettor of outside mechanical manipulation tray interior In, while in being shifted from reagent cabin four without the μ l of DNase, RNase water 0.8 to reagent cabin two, make reaction cumulative volume for 20 μ l, Reacted with the DNA fragmentation reagent mixed liquor in reagent cabin two,
Reaction condition：35-38 DEG C incubates 25-30 minutes,
Then the 0.5 M EDTA that 5 μ l are drawn from reagent cabin three add reagent cabin one, terminate enzymatic reaction,
3rd, DNA fragmentation purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid of the μ l of transferase 12 5 is put into reagent cabin two；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3）The magnetic force rack device of the outside machinery of control rises to the position of reagent cabin two, stands 5-10 minutes, makes magnetic bead and supernatant molten Liquid is separated；
4）The small-sized pipettor of tray interior draws the supernatant in reagent cabin two, is transferred to waste liquid tank；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin two, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in two, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without DNase, RNase water 35-40 μ l to reagent cabin one in, using box Body Internal Small-scale pipettor is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin two, magnetic bead is separated with PCR primer,
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 25 in transfering reagent cabin two in reagent cabin seven, the then nothing in transfering reagent cabin four The μ l of DNase, RNase water 31.5 are inhaled and play 5-10 mixing, with examination up and down to reagent cabin seven using the small-sized pipettor of tray interior Repair reaction and add A reaction mixtures to be reacted in DNA ends in agent cabin seven；
2）Response procedures are as follows：12-15℃ 15-20 min；35-37℃ 15-20 min；70-72℃ 20-25 min；4℃ Keep,
5th, the purifying of end DNA plerosis
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 65 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin seven is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in seven, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 75 to reagent cabin seven in, using small-sized Pipettor is inhaled beat 5-10 times up and down, is stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin seven, magnetic bead is separated with the cDNA of wash-out,
6th, adaptor is connected
μ l of cDNA purification solutions 65 in reagent cabin seven are drawn in reagent cabin eight, is connected with the Adaptor in reagent cabin eight Reaction reagent mixed liquor reacts；
Inhaled up and down using the small-sized pipettor of tray interior and play 5-10 mixing；
Reaction condition：20-25 DEG C incubates 15-20 minutes,
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 83.5 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin eight is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin eight, 30-60 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in eight, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without the μ l of DNase, RNase water 30 to reagent cabin eight in, using box body Internal Small-scale pipettor is inhaled beat 5-10 times up and down, is stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into reagent cabin 8 positions, magnetic bead is separated with the cDNA of wash-out,
8th, PCR amplifications
The μ l of cDNA selective recoveries fragment 23 after the connection adaptor in reagent cabin eight are drawn, are transferred in reagent cabin nine, Reacted with the PCR amplifing reagents mixed liquor in reagent cabin nine,
Response procedures are as follows：95-98 DEG C of 30-45 s of predegeneration；95-98 DEG C of 10-15 s of denaturation；60-65 DEG C of 30- of annealing 40 s；Extend 70-72 DEG C of 30-40 s；6-10 circulation；Extend 70-72 DEG C of 4-5 min eventually,
9th, PCR primer purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the CMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid of the μ l of transferase 45 0 is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin nine is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin nine, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 5-10 minutes is stood, reagent cabin is drawn Supernatant in nine, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, draw in reagent cabin four without DNase, RNase water 35-40 μ l to reagent cabin nine in, using box Body Internal Small-scale pipettor is inhaled beat 5-10 times up and down, is stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead is separated with PCR primer,
10th, PCR primer is taken out
PCR purification solution 25-30 μ l in reagent cabin nine are transferred to library collecting pit, will from outside by library transfer port PCR purifying cDNA solution removes box body, you can take carried out in microarray dataset fasciation into and sequencing, cDNA library can also be deposited In -20 DEG C of preservations.
9. the full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence according to claim 6, its feature It is：When from agent combination two, sequence measurement specifically includes following steps：
Library prepares sample：The single stranded RNA of purifying, is dissolved in ultra-pure water, RNA concentration 150-300 ng/ μ l, RNA purity： OD260/OD280=1.78-2.0,
The reagent held respectively in the 1-9 of reagent cabin,
Library preparation flow is as follows：
Library preparation is carried out with 1 μ g RNA samples, draw 10-15 μ l samples with pipettor is added to sample cell by injection port In, injection port is closed,
1st, RNA reverse transcriptions are cDNA
The RNA samples of 3.3-6.7 μ l in sample cell are transferred to reagent cabin by the small-sized pipettor of outside mechanical manipulation tray interior In one, while shifting Nuclease-free Water 9.3-12.7 μ l from reagent cabin four in reagent cabin one, it is ensured that overall Product is 20 μ l, is reacted with the Reverse Transcription mixed liquor in reagent cabin one, and reaction condition is as follows：37℃ 15 min；85 ℃ 5 seconds；4 DEG C of holdings,
2nd, DNA fragmentationization reaction
15 μ l cDNA solution in sample cell are transferred to reagent cabin two by the small-sized pipettor of outside mechanical manipulation tray interior In, while shifting the μ l of Nuclease-free Water 0.8 from reagent cabin four in reagent cabin two, making the reaction cumulative volume be 20 μ l, react with the DNA fragmentation reagent mixed liquor in reagent cabin two,
Reaction condition：35-38 DEG C incubates 30-40 minutes,
Then the 0.5 M EDTA that 5 μ l are drawn from reagent cabin three add reagent cabin two, terminate enzymatic reaction,
3rd, DNA fragmentation purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 35 μ l is put into reagent cabin two；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
The magnetic force rack device of the outside machinery of control rises to the position of reagent cabin two, stands 5-10 minutes, makes magnetic bead and supernatant solution Separate；
3）The small-sized pipettor of tray interior draws the supernatant in reagent cabin two, is transferred to waste liquid tank；
Magnetic frame is reduced, in the μ l of 80% ethanol 60 in absorption reagent cabin six to reagent cabin two, 30-60 seconds is stood；
4）Magnetic frame is risen into the position of reagent cabin two, 5-10 minutes is stood, magnetic bead and supernatant solution is separated.Draw reagent cabin Supernatant in two, is transferred to waste liquid tank；
5）Repeat step 5,6；
6）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
7）Magnetic frame is reduced, in the Nuclease-free Water 30-35 μ l in absorption reagent cabin four to reagent cabin two, Inhaled up and down using the small-sized pipettor of tray interior and play 5-10 mixing, be stored at room temperature 5-10 minutes；
8）Magnetic frame is risen into the position of reagent cabin two, magnetic bead is separated with the cDNA of wash-out,
4th, DNA ends are repaired reaction and add A reactions：
1）μ l of cDNA purification solutions 20 in transfering reagent cabin two in reagent cabin seven, using on the small-sized pipettor of tray interior 5-10 mixing is played in lower suction, with reagent cabin seven in DNA ends repair reaction and add A reaction mixtures reacted；
Response procedures are as follows：22-25℃ 18-22 min；70-72℃ 20-25 min；4 DEG C of holdings,
5th, the purifying of end DNA plerosis
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 80 μ l is put into reagent cabin seven；
2）The small-sized pipettor of tray interior is inhaled play 5-10 mixing up and down, is stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin seven is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 100 in absorption reagent cabin six to reagent cabin seven, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin seven, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in seven, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 10-15 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 60 in absorption reagent cabin four to reagent cabin seven, profit Inhaled up and down with small-sized pipettor and beaten 5-10 times, be stored at room temperature 5-10 minutes；
Magnetic frame is risen into the position of reagent cabin seven, magnetic bead is separated with the DNA of wash-out,
6th, adaptor is connected
μ l of cDNA purification solutions 50 in reagent cabin seven are drawn in reagent cabin eight, is connected with the Adaptor in reagent cabin eight Reaction reagent mixed liquor reacts；
Inhaled up and down using the small-sized pipettor of tray interior and play 10-15 mixing；
Reaction condition：22-25 DEG C incubates 15-20 minutes,
7th, the DNA fragmentation selective recovery of adaptor is connected
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 60 μ l is put into reagent cabin eight；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin eight is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin eight, 30-60 seconds is stood；
6）Magnetic frame is risen into reagent cabin 8 positions, 5-10 minutes is stood, magnetic bead and supernatant solution is separated, reagent cabin is drawn Supernatant in eight, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 5-10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the μ l of Nuclease-free Water 30 in absorption reagent cabin four to reagent cabin eight, profit Inhaled up and down with the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into reagent cabin 8 positions, magnetic bead is separated with the cDNA of wash-out,
8th, PCR amplifications
The μ l of cDNA selective recoveries fragment 20 after the connection adaptor in reagent cabin eight are drawn, are transferred in reagent cabin nine, Reacted with the PCR amplifing reagents mixed liquor in reagent cabin nine,
Response procedures are as follows：95-98 DEG C of 2-2.5 min of predegeneration；95-98 DEG C of 30-40 s of denaturation；60-65 DEG C of annealing 30-40 s；Extend 70-72 DEG C of 60-90 s；4-10 circulation；Extend 70-72 DEG C of 4-5 min eventually,
9th, PCR primer purifying
1）Inhale to beat 10-15 times up and down by the small-sized pipettor of tray interior and thoroughly mix the AMPure magnetic beads in reagent cabin five, Then the magnetic bead liquid for shifting 40 μ l is put into reagent cabin nine；
2）Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
3）Magnetic frame is risen into the position of reagent cabin nine, 5-10 minutes is stood, magnetic bead and supernatant solution is separated；
4）The supernatant in reagent cabin nine is drawn, waste liquid tank is transferred to；
5）Magnetic frame is reduced, in the μ l of 80% ethanol 200 in absorption reagent cabin six to reagent cabin nine, 30-60 seconds is stood；
6）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead and supernatant solution is separated, 5-10 minutes is stood, reagent cabin is drawn Supernatant in nine, is transferred to waste liquid tank；
7）Repeat step 5,6；
8）It is stored at room temperature 5-10 minutes, magnetic bead is dried in atmosphere；
9）Magnetic frame is reduced, in the Nuclease-free Water 35-40 μ l in absorption reagent cabin four to reagent cabin nine, Inhaled up and down using the small-sized pipettor of tray interior and beaten 5-10 times, be stored at room temperature 5-10 minutes；
10）Magnetic frame is risen into the position of reagent cabin nine, magnetic bead is separated with PCR primer,
10th, PCR primer is taken out
PCR purification solution 25-30 μ l in reagent cabin nine are transferred to library collecting pit, shifting hole by library will from outside PCR purification solutions remove box body, you can take carried out in microarray dataset fasciation into and sequencing, also RNA libraries can be stored in -20 DEG C preserve.
10. the full-automatic RNA libraries preparation facilities for being applied to second generation high-flux sequence according to claim 7, it is special Levy and be：Sequence measurement specifically also includes step：
Library Quality is detected：
Fragment length distribution in the RNA libraries agarose gel electrophoresis detection RNA libraries for preparing, if the text for building Storehouse quality is good, can be directly sequenced.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725905A (en) * 2020-12-31 2021-04-30 张研 Digital microfluidic library construction system
WO2024056012A1 (en) * 2022-09-15 2024-03-21 上海思路迪生物医学科技有限公司 Automatic ngs library preparation and sub-packaging kit and library construction method

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101802164A (en) * 2007-07-13 2010-08-11 汉迪实验室公司 Intergrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
CN103103624A (en) * 2011-11-15 2013-05-15 深圳华大基因科技有限公司 Method for establishing high-throughput sequencing library and application thereof
CN103725591A (en) * 2010-07-23 2014-04-16 贝克曼考尔特公司 Cartridge loading unit
CN104145014A (en) * 2011-11-11 2014-11-12 爱库倍特公司 Systems and methods for performing amplicon rescue multiplex polymerase chain reaction (PCR)
CN104152349A (en) * 2014-07-29 2014-11-19 山东艾克韦生物技术有限公司 Automatic detection reagent cassette

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101802164A (en) * 2007-07-13 2010-08-11 汉迪实验室公司 Intergrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
CN103725591A (en) * 2010-07-23 2014-04-16 贝克曼考尔特公司 Cartridge loading unit
CN104145014A (en) * 2011-11-11 2014-11-12 爱库倍特公司 Systems and methods for performing amplicon rescue multiplex polymerase chain reaction (PCR)
CN103103624A (en) * 2011-11-15 2013-05-15 深圳华大基因科技有限公司 Method for establishing high-throughput sequencing library and application thereof
CN104152349A (en) * 2014-07-29 2014-11-19 山东艾克韦生物技术有限公司 Automatic detection reagent cassette

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725905A (en) * 2020-12-31 2021-04-30 张研 Digital microfluidic library construction system
WO2024056012A1 (en) * 2022-09-15 2024-03-21 上海思路迪生物医学科技有限公司 Automatic ngs library preparation and sub-packaging kit and library construction method

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