CN108130323A - A kind of banking process suitable for the analysis of plant transposase accessibility chromatin - Google Patents

A kind of banking process suitable for the analysis of plant transposase accessibility chromatin Download PDF

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CN108130323A
CN108130323A CN201711386040.4A CN201711386040A CN108130323A CN 108130323 A CN108130323 A CN 108130323A CN 201711386040 A CN201711386040 A CN 201711386040A CN 108130323 A CN108130323 A CN 108130323A
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library
nucleus
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banking process
follows
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童云广
王华印
徐鹭芹
赵楠
胡浅浅
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Oing (hangzhou) Gene Technology Co Ltd
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Abstract

The invention discloses a kind of banking process suitable for the analysis of plant transposase accessibility chromatin, pre-treatment is carried out to plant tender tissue first, it is filtered after lysed cells wall, utilize DAPI dyeing separation and collection nucleus on flow cytometer, filtration cell core effectively reduces the interference of cell wall and subcellular organelle to experimental data.Carry out Tn5 swivel bases endonuclease reaction and purifying, qPCR determines to build recurring number needed for library, equimolar mixing finally is carried out to obtained single library, it sorts to obtain the upper machine library of high quality using the two-wheeled magnetic bead ratio optimized, studying the accessible transposase nuclear chromatin high-flux sequence of plant tender tissue for numerous scientific research personnel provides important experimental method reference.

Description

A kind of banking process suitable for the analysis of plant transposase accessibility chromatin
Technical field
The present invention relates to technical field of molecular biology, more particularly to a kind of plant transposase accessibility that is suitable for dyes The banking process of matter analysis.
Background technology
ATAC-seq is that transposase accessibility nuclear chromatin region is analyzed by using high-flux sequence (Assay for Transposase-Accessible Chromatin with high throughput sequencing) A kind of innovation epigenetics investigative technique.The technology is by transposase to nuclear chromatin area open under certain specific space-time Domain is cut, and then obtains the regulating and controlling sequence of all active transcriptions in genome under the specific space-time.
Eukaryocyte is by genomic DNA and histone by the assembled foldings of different levels into chromatin, these accurate groups Dress information plays a decisive role in gene transcription regulation.The accessibility of Chromatin domains be specific trans-acting factor and The premise of cis-regulating element interaction, the change of the exception or the chromatin control factor of gene expression all will be to cell Destiny generates far-reaching influence, and then leads to the generation and development of various diseases.The technology has been widely used in transcribing at present The identification of regulating and controlling sequence.ATAC-seq technologies can operate with the research of all eukaryocyte reprogrammings, be great in medical domain The tool of new generation of the researchs such as disease incidence mechanism, mechanism of drug action, new drug development and biomarker function.
Chromatin Structure plays key effect in promotion gene expression control aspect.The cis element that transcription factor combines leads to It is often related to the accessibility of Chromatin domains.Therefore, these accessibility regions are identified in the plant genome, are beneficial to Improve the understanding of the relationship between transcription factor combination, chromatin state and gene expression regulation three.ATAC-seq leads to now It is commonly used for systematically identifying the cis-regulatory region in Animal genome and DNA footprints, however, this method is to plant species Application be one challenge.In plant, the presence of cell wall and organelle, the shortage of stable cell lines prevent the technology to exist Application in plant.Often containing organelle genes groups such as mitochondrial and chloroplasets in plant dyeing matter extract, due to organelle Genome can also be approached and cut by Tn5 transposases, therefore reduce the Tn5 swivel base enzyme activity for cleaving plant genome Property.
Invention content
The purpose of the present invention is to provide it is a kind of suitable for plant transposase accessibility chromatin analysis banking process, The experiment interference that genome is brought in plant leaf blade cell wall and subcellular organelle is reduced, using the experiment condition optimized, is led to Two-wheeled magnetic bead sorting is crossed, retains most preferably upper machine sequencing library size, so as to obtain machine data in high quality, is easily connect for plant leaf blade Nearly transposase nuclear staining Quality Research provides important experimental method reference.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of banking process suitable for the analysis of plant transposase accessibility chromatin includes the following steps：
S1：Plant tissue (tender tissue) sample obtains nucleus suspension after enzymolysis liquid TS2 and lysate TS1 processing Liquid A；
S2：(DAPI dyeing), sorting are dyed to the nucleus in nuclei suspension A, obtains nucleus B；
S3：Swivel base reaction and purifying are carried out to nucleus B, obtains swivel base product C；
S4：PCR amplification is carried out to swivel base product C, obtains amplified production D；The sampling amount of swivel base product C is 1~100ng, It is preferred that sampling amount is 40~60ng；
S5：Part amplified production D is taken to carry out qPCR with determine it is additional needed for PCR cycle number, according to it is determining it is additional needed for PCR cycle number continues to expand to remaining amplified production D, obtains amplified production E；
S6：Purifying amplified production E obtains single library, and single library carries out segment sorting, obtains machine library.
Preferably, S1 is as follows：200~300mg plant tissues are taken, culture on ice is being positioned over blade Tissue is chopped into homogenate shape in ware, adds in 2.5~3.0mL enzymolysis liquids TS2 enzymolysis 4h removal cell walls；4 DEG C, 300~ Supernatant is abandoned after 400rpm centrifugations 10min；The lysate TS1 of 2.5~3.0mL precoolings is added in, is transferred in centrifuge tube and is placed in mixing 100~150rpm vibrates 5min on device, obtains the mixed liquor after cracking completely；By mixed liquor filtering twice, gained filtrate distribution In the layering liquid of different densities, 1000~1300rpm centrifuges 20~25min, collects the nucleus of separation, adds in 600 μ L and splits Liquid TS1 is solved, obtains nuclei suspension A.
It is as follows to be layered liquid configuration method：The layering stoste of 9 times of volumes is taken, 10 × phosphate buffer of 1 times of volume is added in, obtains To 100% layering liquid, 2.5mL 100% is taken to be layered liquid, adds in 1 × phosphate buffers of 2.5mL, obtain 50% layering liquid, The layering liquid of its density is prepared successively using 100% layering liquid and 1 × phosphate buffer；The preparation method of 10 × phosphate buffer It is as follows：1.37M NaCl, 20.7mM KCl, 100mM Na2HPO4·12H2O, 17.6mM KH2PO4；1 × phosphate buffer is matched Method processed is as follows：The ultra-pure water of 9 times of volumes adds in 10 × phosphate buffer of 1 times of volume, is uniformly mixed.
Preferably, S2 is as follows：Nuclei suspension A using 40 μm of sterilizing filters is filtered, collects filter Liquid carries out nuclear targeting and is sorted according to nucleus size, and the nucleus use of sorting is previously charged into 600 μ L lysates The centrifuge tube of TS1 is collected；After the cell nuclear mass that micro- Microscopic observation sorts, by 4 DEG C of nucleus being collected into, 1000~1300rpm centrifuges 15min, abandons supernatant, adds in 600 μ L washing buffers washing nucleus, 1000~1300rpm centrifugations Supernatant is abandoned after 10min and obtains nucleus B.
Preferably, the washing buffer component composition is as follows：10mM Tris-HCl pH8.0,5mM MgCl2。
Preferably, S3 is as follows：Nucleus B is taken to carry out swivel base reaction immediately, nucleus B adds in Tn5 swivel bases After enzyme, 37 DEG C of 30~70min of water-bath；Swivel base product after water-bath is purified, obtains swivel base product C.In this step Nucleus amount and digestion time are the key factors for influencing Library Quality, are obtained according to experimental result, 40000 nucleus, Digestion time 50min, can obtain preferable experimental result, and nucleus is excessive, and the digestion time is too short, and chromatin can be caused inabundant Digestion, segment is larger, influences machine cluster；Nucleus is very little, and the digestion time is long, can lead to excessive digestion, digestion heterochromatin Region.
Preferably, the enzymolysis liquid TS2 components composition is as follows：0.5%~1.5% cellulase, 0.3%~0.6% Pectase, 0.3%~0.6% hemicellulase, 0.45~0.55M mannitol, the 2- (N- of 20mM KCl, 10mM, pH5.7 Quinoline) ethanesulfonic acid monohydrate, 10mM CaCl2, 0.1% bovine serum albumin(BSA).The preparation method of enzymolysis liquid TS2 is as follows：Mixing Cellulase, pectase, hemicellulase, mannitol, KCl, 2- (N- morpholines) ethanesulfonic acid monohydrate are placed in 56 DEG C of water-baths 10min after being cooled to room temperature, adds in CaCl2And bovine serum albumin(BSA), and in 0.45 μm of membrane filtration to culture dish.
Preferably, the lysate TS1 components composition is as follows：20mM Tris-HCl pH 8.0,75mM KCl, 15mM NaCl, 0.5mM spermine, 10mM beta -mercaptoethanols, 2%PVP, 0.3%TritonX-100.
Preferably, determining additional required PCR cycle number in S5, calculation is as follows：According to quantitative fluorescent PCR curve Figure, corresponding recurring number is additional required recurring number when taking maximum fluorescence value 1/4.The numerical value of the recurring number is 0~7, preferably 4 ~6 cycles.
Preferably, S6 is as follows：By amplified production E respectively after purification, single library is obtained, measures single text Multiple single library equimolar concentrations are mixed into mixing library by the concentration in library according to concentration, and mixing library uses two-wheeled DNA magnetic Pearl carry out segment sorting, retain 200~1000bp (preferably 200~700bp) DNA fragmentation sorted after library, use core Acid analysis instrument carries out Library Quality detection, and machine library is obtained after detection is qualified.
Preferably, mixing library carries out segment sorting using two-wheeled DNA magnetic beads, the volume ratio of first round magnetic bead is 0.4 ×~0.9 ×, the volume ratio of the second wheel magnetic bead for 0.5 ×~1.0 ×.Magnetic bead volume ratio × meaning be：DNA magnetic Pearl and the volume ratio of amplified production E.Preferred separation condition be first round magnetic bead volume ratio be 0.5 ×, the second wheel magnetic bead body Product ratio for 0.7 ×.
The beneficial effects of the invention are as follows：The present invention carries out pre-treatment to plant tender tissue first, mistake after lysed cells wall Filter effectively reduces cell wall and Asia using DAPI dyeing separation and collection nucleus, filtration cell core on flow cytometer Interference of the organelle to experimental data.Tn5 swivel bases endonuclease reaction and purifying are carried out, qPCR determines to build recurring number needed for library, finally Equimolar mixing is carried out to obtained single library, sorts to obtain the upper machine text of high quality using the two-wheeled magnetic bead ratio optimized Library studies the accessible transposase nuclear chromatin high-flux sequence of plant tender tissue for numerous scientific research personnel and provides important experiment Method refers to.
Description of the drawings
Fig. 1 is the method flow schematic diagram of the present invention.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention is described in further detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art. Method in following embodiments is the conventional method of this field unless otherwise instructed.
Total embodiment：
A kind of banking process suitable for the analysis of plant transposase accessibility chromatin includes the following steps：
S1：Plant tissue sample obtains nuclei suspension A after enzymolysis liquid TS2 and lysate TS1 processing；
S2：Nucleus in nuclei suspension A is dyed, is sorted, obtains nucleus B；
S3：Swivel base reaction and purifying are carried out to nucleus B, obtains swivel base product C；
S4：PCR amplification is carried out to swivel base product C, obtains amplified production D；
S5：Part amplified production D is taken to carry out qPCR with determine it is additional needed for PCR cycle number, according to it is determining it is additional needed for PCR cycle number continues to expand to remaining amplified production D, obtains amplified production E；
S6：Purifying amplified production E obtains single library, and single library carries out segment sorting, obtains machine library.
The enzymolysis liquid TS2 components form as follows：0.5%~1.5% cellulase, 0.3%~0.6% pectase, 0.3%~0.6% hemicellulase, 0.45~0.55M mannitol, 2- (N- morpholines) second sulphur of 20mM KCl, 10mM, pH5.7 Sour monohydrate, 10mM CaCl2, 0.1% bovine serum albumin(BSA).
The lysate TS1 components form as follows：20mM Tris-HCl pH 8.0,75mM KCl, 15mM NaCl, 0.5mM spermine, 10mM beta -mercaptoethanols, 2%PVP, 0.3%TritonX-100.
It applies the technical scheme of the present invention, during using lysate cracking plant cell, can leniently destroy plant The cell membrane of object cell is discharged on the basis of not damaging cells core.Using DAPI to nuclear targeting and then according to it Size is sorted, and can largely reduce the influence of DNA in the organelles such as plant cell wall and mitochondria, chloroplaset. Utilize Tn5 transposases, on the one hand, for the technologies such as MNase-seq, required cell number is few, on the other hand, Tn5 Transposase can be that the segment of well cutting adds joint sequence, save experiment and taken while chromatin active regions are cut Between.The problems such as additional required recurring number being determined using qPCR, being avoided because of the PCR Preferences introduced and amplification redundancy.It utilizes Two-wheeled magnetic bead sorting can retain machine and best library size is sequenced, so as to obtain machine data in high quality.The present invention utilizes a variety of Technological means provides important experimental method reference for the accessible transposase nuclear staining Quality Research of plant leaf blade.
Preferably, S1 includes weighing for plant tissue, the homogenate of tissue, the cracking of cell wall, the cracking of cell membrane, cell The filtering and purifying of core.
Preferably, S2 includes the filtering of nucleus suspension, and the DAPI of nucleus is dyed, the sorting of nucleus, nucleus Washing.
Preferably, S3 includes chromatinic Tn5 swivel bases enzymatic treatment, the purifying of digestion products.
Preferably, S4 and S5 include purifying DNA PCR, additional cycles number needed for PCR determine, remaining PCR product after Continuous amplification.
Preferably, S6 includes the purifying of PCR product, the segment screening of purified product, and library concentration detects, Library Quality Bioanalyzer is detected, the high-flux sequence in library, the bioinformatic analysis of data.
Embodiment 1：
A kind of banking process suitable for the analysis of tree peony floral leaf transposase accessibility chromatin, the flow chart of reference Fig. 1, Specific implementation step is as follows：
(1) separation of nucleus.
A. 200mg tree peony floral leaves are weighed.
B. tissue is chopped into homogenate shape in culture dish on ice is positioned over sharp blade.
C. 2.5mL TS2 enzymolysis liquids enzymolysis 4h is added in remove cell wall.TS2 enzymolysis liquids are formulated as：0.5% cellulose Enzyme, 0.4% pectase, 0.3% hemicellulase, 0.5M mannitol, 20mmol/L KCl, 10mmol/L MES (2- (N- Quinoline) ethanesulfonic acid monohydrate, pH 5.7) mixed liquor is placed in 56 DEG C of water-bath 10min, after being cooled to room temperature, add in 10mmol/L CaCl2, 0.1%BSA, and with 0.45 μm of membrane filtration.
D. after digesting, 4 DEG C, 400rpm centrifugation 10min discard supernatant liquid.
E. precipitation adds in the TS1 lysates of 2.5mL precoolings, after pipettor blows and beats resuspension repeatedly, is transferred to juxtaposition in centrifuge tube 5min is vibrated in 100~150rpm on vortex mixer.The composition of TS1 lysates is：20mM Tris-HCl pH 8.0,75mM KCl, 15mM NaCl, 0.5mM spermine, 10mM beta -mercaptoethanols, 2%PVP, 0.3%TritonX-100.
F. completely after cracking, with filter-cloth filtering twice, filtrate is collected.
G. filtrate is layered liquid gradient centrifugation, 400~1300rpm centrifugations with density 50%, 45%, 37.5%, 35% 20min。
H. the nucleus of separation is collected, 600 μ L TS1 lysates is added to be resuspended.
(2) the DAPI dyeing and sorting of nucleus.
A. nucleus suspension is filtered using 40 μm of sterilizing filter.
B. filtrate is collected to be dyed with DAPI.
C. it using flow cytometer, is sorted according to the size of nucleus, collects 40000 nucleus.
D. the nucleus centrifugation collected, 4 DEG C, 1300rpm centrifugation 15min, discards supernatant.
E. precipitation is washed with washing buffer.The composition of washing buffer is：10mM Tris-HCl pH8.0,5mM MgCl2。
(3) Tn5 swivel bases enzyme reaction.
A. swivel base enzyme modification.Transposase is coated with joint sequence：
Adapter1：5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’(SEQ ID No.1)
3 '-TCTACACATATTCTCTGTC-5 ' (SEQ ID No.2),
Adapter2：5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’(SEQ ID No.3)
3’-TCTACACATATTCTCTGTC-5’(SEQ ID No.4)。
B. the Tn5 transposases and buffer solution added in after modification carries out swivel base reaction.Take 10 μ 5 × TTBL of L Buffer (TruePrep Tagment Buffer L Nanjing Vazyme Biotechnology Co., Ltd.), 5 μ L Tn5 transposases, 50ng DNA, Pipettor gently blows and beats of short duration centrifugation after mixing, 37 DEG C of water-bath 50min.
(4) digestion products purify
A. after the completion of swivel base reaction, with Qiagen MinElute kit, (full name is QIAGEN MinElute PCR Purification Kit, Cat No.28204) it is purified.
B. the Buffer PB (plus pH indicator I, commercially available) of 5 times of volumes are added in digestion products, are mixed evenly.
C. mixed liquor is transferred to MinElute column (commercially available), 13000rpm centrifuges 1min at room temperature.
D. filtrate is abandoned, column (column) is put back in 2mL collecting pipes, has added in 750 μ L Buffer PE (plus anhydrous second Alcohol), 13000rpm centrifuges 1min at room temperature.
E. filtrate is abandoned, column is put back in 2mL collecting pipes, 13000rpm skies centrifugation 2min.
F. it shifts in column to clean 1.5mL centrifuge tubes, opens lid, drying at room temperature 3min.
G. 26 μ L Nuclease free water (nuclease-free water) are added in column center membranes, be placed at room temperature for 2min, 13000rpm centrifugation 2min collect filtrate.
(5) PCR is enriched with
A. following mixed liquor is prepared on ice：
B. rear brief centrifugation is mixed evenly, in being reacted as follows on qPCR instrument：
C. each primer sequence is as follows：
Custom Primer 1：
5’-AATGATACGGCGACCACCGAGATCTACAC index TCGTCGGCAGCGTCAGATGT-3’(SEQ ID No.5)
Custom Primer 2：
5’-CAAGCAGAAGACGGCATACGAGAT index GTCTCGTGGGCTCGGAGATG-3’(SEQ ID No.6)
P5：
5’-AATGATACGGCGACCACCGAGATCTACAC-3’(SEQ ID No.7)
P7：
5’-CAAGCAGAAGACGGCATACGAGAT-3’(SEQ ID No.8)。
(6) qPCR determines additional cycles number
A. following mixed liquor is prepared on ice, 5 μ L PCR products is taken to be reacted, resultant product is placed on ice：
B. rear brief centrifugation is mixed evenly, in being reacted as follows on qPCR instrument：
C. the recurring number according to needed for the calculating of qPCR results is additional.Computational methods, which are that 1/4 place of maximum fluorescence value is corresponding, to be followed Number of rings.According to result of calculation, the additional recurring number needed of counting is 7cycles, and remaining sample is put into PCR instrument and is continued instead It should.Conversely, it then needs to optimize adjustment to sample initial amount and digestion time.
(7) PCR product purifies
A.PCR products are purified with Qiagen MinElute kit.
The Buffer PB (plus pH indicator I) of 5 times of volumes are added in b.PCR products, are mixed evenly.
C. mixed liquor is transferred to MinElute column, 13000rpm centrifuges 1min at room temperature.
D. filtrate is abandoned, column is put back in 2mL collecting pipes, adds in 750 μ L Buffer PE (plus absolute ethyl alcohol), room The lower 13000rpm centrifugations 1min of temperature.
E. filtrate is abandoned, column is put back in 2mL collecting pipes, 13000rpm skies centrifugation 2min.
F. it shifts in column to clean 1.5mL centrifuge tubes, opens lid, drying at room temperature 3min.
G. 22 μ L Nuclease free water are added in column center membranes, are placed at room temperature for 2min, 13000rpm from Heart 2min collects filtrate.
(8) Library Quality assessment 1：Agilent 2100
A. kit (DNA dye concentrate, DNA gel matrix) equilibrium at room temperature 30min.
B. vortex DNA dye concentrate 10sec draw 25 μ L dyestuffs and are added to DNA gel after centrifugation Fully be vortexed mixing in matrix, wink from.
C. it shifts in mixed liquor to spin filter (solvent resistant column), ± 20% room temperatures of 2240g centrifugation 15min.
D. 4 DEG C of the glue dye centrifuged is kept in dark place.
E. syringe piston is placed at 1.0mL by adjustment glue pressing device cartridge clip in C gears (last step).
F. a new DNA1000 chip is taken out, 9 μ L gel-dye mix are added in the hole labeled as G, and (liquid-transfering gun falls It inhales).
G. chip being placed in priming station and being shut (can hear when priming station are correctly closed " card " sound).
H. injection ram is slowly pressed downward until being blocked by cartridge clip.60sec is kept, discharges cartridge clip, piston rebounds automatically (piston should bounce back into the position of 0.7mL immediately, otherwise possible priming station gas leakage).
I. piston is slowly retracted into 1.0mL positions, opens glue pressing device and take out detection chip.
J. it is marked in the hole having to remaining 2 and adds in 9.0 μ L gel-dye mix (liquid-transfering gun suck-back).
K. in 12 sample wells and enter 5 μ L DNA Marker (green) in 1 Ladder hole.
L. the 1 processed Ladder of μ L are added in Ladder holes, and (Ladder needs to dispense, often 1.1 μ L of pipe, -80 DEG C of guarantors It deposits).
M. 1 μ L samples are added in each sample well, 1 μ L Nuclease free are added in not used hole water。
N. chip mixing is centrifuged into 1min, the chip prepared needs machine testing in 5min, needs to determine before detection every There is no bubble in a hole.
Data result is as follows：
(9) library fragments screen
A. VAHTS DNA Clean Beads are shifted to an earlier date into 30min from 2-8 DEG C of taking-up, balanced to room temperature, reverse or vortex shakes Swinging makes the abundant mixing of magnetic bead.
B. the magnetic bead of 0.5 × volume (25 μ L), pipettor piping and druming mixing are added according to elution liquor capacity.
C. 5min is incubated at room temperature, DNA is made to be attached on magnetic bead.
D. sample is placed in 5min on magnetic frame, after solution clarification, carefully takes supernatant in new EP pipes.
E. the magnetic bead of 0.7 × volume (35 μ L) of elution liquor capacity, pipettor piping and druming mixing are added in pipe.
F. 5min is incubated at room temperature, sample is placed on magnetic frame, after solution clarification, carefully removes supernatant.
G. sample is kept to be placed on magnetic frame always, adds in the 80% ethyl alcohol rinsing magnetic bead of the 200 fresh configurations of μ L, room temperature 30sec is incubated, carefully removes supernatant.
H. it repeats to be rinsed with alcohol primary.
I. keep sample always on magnetic frame, drying at room temperature of uncapping magnetic bead 3min.
J. sample from magnetic frame is taken off, adds in suitable Nuclease free water.Pipettor piping and druming is mixed It is even, it is incubated at room temperature 5min.
K. sample is placed in 2min on magnetic frame, careful to shift in supernatant to new centrifuge tube after solution clarification.
(10) Library Quality assessment 2：Agilent 2100
A. kit (DNA dye concentrate, DNA gel matrix) equilibrium at room temperature 30min.
B. vortex DNA dye concentrate 10sec draw 25 μ L dyestuffs and are added to DNA gel after centrifugation Fully be vortexed mixing in matrix, wink from.
C. it shifts in mixed liquor to spin filter, ± 20% room temperatures of 2240g centrifugation 15min.
D. 4 DEG C of the glue dye centrifuged is kept in dark place.
E. syringe piston is placed at 1.0mL by adjustment glue pressing device cartridge clip in C gears (last step).
F. a new DNA1000 chip is taken out, 9 μ L gel-dye mix are added in the hole labeled as G, and (liquid-transfering gun falls It inhales).
G. chip being placed in priming station and being shut (can hear when priming station are correctly closed " click " sound).
H. injection ram is slowly pressed downward until being blocked by cartridge clip.60sec is kept, discharges cartridge clip, piston rebounds automatically (piston should bounce back into the position of 0.7mL immediately, otherwise possible priming station gas leakage).
I. piston is slowly retracted into 1.0mL positions, opens glue pressing device and take out detection chip.
J. it is marked in the hole having to remaining 2 and adds in 9.0 μ L gel-dye mix (liquid-transfering gun suck-back).
K. in 12 sample wells and enter 5 μ L DNA Marker (green) in 1 Ladder hole.
L. the 1 processed Ladder of μ L are added in Ladder holes, and (Ladder needs to dispense, often 1.1 μ L of pipe, -80 DEG C of guarantors It deposits).
M. 1 μ L samples are added in each sample well, 1 μ L Nuclease free are added in not used hole water。
N. chip mixing is centrifuged into 1min, the chip prepared needs machine testing in 5min, needs to determine before detection
There is no bubble in each hole.
(11) machine on library
Library concentration according to obtained by step (10) is 4.68ng/ μ L, and library mean size is 294bp, and molar concentration is 24.49nM, by library be diluted to it is upper it is confidential ask after, be sequenced using Nextseq500 PE150 strategies, as a result given birth to Object information analysis.
Embodiment 2：A kind of banking process suitable for the analysis of tobacco leaf transposase accessibility chromatin, with reference to Fig. 1 Flow chart, specific implementation step is as follows：
(1) separation of nucleus.
A. 200mg tobacco leafs are weighed.
B. tissue is chopped into homogenate shape in culture dish on ice is positioned over sharp blade.
C. 2.5mL TS2 enzymolysis liquids enzymolysis 4h instrument removal cell walls are added in.TS2 enzymolysis liquids are formulated as：1.3% cellulose Enzyme, 0.4% macerozyme, 0.3% pectase, 0.4M mannitol (pH 5.7) mixed liquor are placed in 56 DEG C of water-bath 10min, are cooled to room Wen Hou adds in 10mmol/L CaCl2, 0.1%BSA, and with 0.45 μm of membrane filtration.
D. after digesting, 4 DEG C, 400rpm centrifugation 10min discard supernatant liquid.
E. precipitation adds in 2.5mL precooling TS1 lysates, after pipettor blows and beats resuspension repeatedly, is transferred in centrifuge tube and is placed in 100~150rpm vibrates 5min on vortex mixer.The composition of TS1 lysates is：20mM Tris-HCl pH 8.0,75mM KCl, 15mM NaCl, 0.5mM spermine, 10mM beta -mercaptoethanols, 2%PVP, 0.3%TritonX-100.
F. completely after cracking, with magical filter-cloth filtering twice, filtrate is collected.
G. filtrate is layered liquid gradient centrifugation, 400~1300rpm centrifugations 25min with density 50%, 45%, 40%, 35%.
H. the nucleus of separation is collected, 600 μ L TS1 lysates is added to be resuspended.
(2) the DAPI dyeing and sorting of nucleus.
A. nucleus suspension is filtered using 40 μm of sterilizing filter.
B. filtrate is collected to be dyed with DAPI.
C. it using flow cytometer, is sorted according to the size of nucleus, collects 40000 nucleus.
D. the nucleus centrifugation collected, 4 DEG C, 1300rpm centrifugation 15min are discarded supernatant.
E. precipitation is washed with washing buffer.The composition of washing buffer is：10mM Tris-HCl pH8.0,5mM MgCl2。
(3) Tn5 swivel bases enzyme reaction.
A. swivel base enzyme modification.Transposase is coated with joint sequence：
Adapter1：5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’
3’-TCTACACATATTCTCTGTC-5’
Adapter2：5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’
3’-TCTACACATATTCTCTGTC-5’。
B. the Tn5 transposases and buffer solution added in after modification carries out swivel base reaction.Take 10 μ 5 × TTBL of L Buffer, 5 μ L Tn5 transposases, 50ng DNA, pipettor gently blow and beat of short duration centrifugation after mixing, 37 DEG C of water-bath 60min.
(4) digestion products purify
A. after the completion of swivel base reaction, purified with Qiagen MinElute kit.
B. the Buffer PB (plus pH indicator I) of 5 times of volumes are added in digestion products, are mixed evenly.
C. mixed liquor is transferred to MinElute column, 13000rpm centrifuges 1min at room temperature.
D. filtrate is abandoned, column is put back in 2mL collecting pipes, adds in 750 μ L Buffer PE (plus absolute ethyl alcohol), room The lower 13000rpm centrifugations 1min of temperature.
E. filtrate is abandoned, column is put back in 2mL collecting pipes, 13000rpm skies centrifugation 2min.
F. it shifts in column to clean 1.5mL centrifuge tubes, opens lid, drying at room temperature 3min.
G. 26 μ L Nuclease free water are added in column center membranes, are placed at room temperature for 2min, 13000rpm from Heart 2min collects filtrate.
(5) PCR is enriched with
A. prepare following mixed liquor on ice (with embodiment 1)：
B. rear brief centrifugation is mixed evenly, in being reacted as follows on qPCR instrument：
C. each primer sequence is the same as embodiment 1.
(6) quantitative fluorescent PCR determines additional cycles number
A. following mixed liquor is prepared on ice, 5 μ L PCR products is taken to be reacted, resultant product is placed on ice：
B. rear brief centrifugation is mixed evenly, in being reacted as follows on qPCR instrument：
C. the recurring number according to needed for fluorescent quantitative PCR result calculating is additional.Computational methods are right at maximum fluorescence value 1/4 The recurring number answered.According to result of calculation, additional required recurring number is 6cycles, and remaining sample is put into PCR instrument and is continued Reaction.Conversely, it then needs to optimize adjustment to sample initial amount and digestion time.
(7) PCR product purifies
A.PCR products are purified with Qiagen MinElute kit.
The Buffer PB (plus pH indicator I) of 5 times of volumes are added in b.PCR products, are mixed evenly.
C. mixed liquor is transferred to MinElute column, 13000rpm centrifuges 1min at room temperature.
D. filtrate is abandoned, column is put back in 2mL collecting pipes, adds in 750 μ L Buffer PE (plus absolute ethyl alcohol), room The lower 13000rpm centrifugations 1min of temperature.
E. filtrate is abandoned, column is put back in 2mL collecting pipes, 13000rpm skies centrifugation 2min.
F. it shifts in column to clean 1.5mL centrifuge tubes, opens lid, drying at room temperature 3min.
G. 22 μ L Nuclease free water are added in column center membranes, are placed at room temperature for 2min, 13000rpm from Heart 2min collects filtrate.
(8) Library Quality assessment 1：Agilent 2100
A. kit (DNA dye concentrate, DNA gel matrix) equilibrium at room temperature 30min.
B. vortex DNA dye concentrate 10sec draw 25 μ L dyestuffs and are added to DNA gel after centrifugation Fully be vortexed mixing in matrix, wink from.
C. it shifts in mixed liquor to spin filter, ± 20% room temperatures of 2240g centrifugation 15min.
D. 4 DEG C of the glue dye centrifuged is kept in dark place.
E. syringe piston is placed at 1.0mL by adjustment glue pressing device cartridge clip in C gears (last step).
F. a new DNA1000 chip is taken out, 9 μ L gel-dye mix are added in the hole labeled as G, and (liquid-transfering gun falls It inhales).
G. chip being placed in priming station and being shut (can hear when priming station are correctly closed " card " sound).
H. injection ram is slowly pressed downward until being blocked by cartridge clip.60sec is kept, discharges cartridge clip, piston rebounds automatically (piston should bounce back into the position of 0.7mL immediately, otherwise possible priming station gas leakage).
I. piston is slowly retracted into 1.0mL positions, opens glue pressing device and take out detection chip.
J. it is marked in the hole having to remaining 2 and adds in 9.0 μ L gel-dye mix (liquid-transfering gun suck-back).
K. in 12 sample wells and enter 5 μ L DNA Marker (green) in 1 Ladder hole.
L. the 1 processed Ladder of μ L are added in Ladder holes, and (Ladder needs to dispense, often 1.1 μ L of pipe, -80 DEG C of guarantors It deposits).
M. 1 μ L samples are added in each sample well, 1 μ L Nuclease free are added in not used hole water。
N. chip mixing is centrifuged into 1min, the chip prepared needs machine testing in 5min, needs to determine before detection every There is no bubble in a hole.
Data result is as follows：
(9) library fragments screen
A. VAHTS DNA Clean Beads are shifted to an earlier date into 30min from 2-8 DEG C of taking-up, balanced to room temperature, reverse or vortex shakes Swinging makes the abundant mixing of magnetic bead.
B. the magnetic bead of 0.5 × volume (23 μ L), pipettor piping and druming mixing are added according to elution liquor capacity.
C. 5min is incubated at room temperature, DNA is made to be attached on magnetic bead.
D. sample is placed in 5min on magnetic frame, after solution clarification, carefully takes supernatant in new EP pipes.
E. the magnetic bead of 0.7 × volume (32 μ L) of elution liquor capacity, pipettor piping and druming mixing are added in pipe.
F. 5min is incubated at room temperature, sample is placed on magnetic frame, after solution clarification, carefully removes supernatant.
G. sample is kept to be placed on magnetic frame always, adds in the 80% ethyl alcohol rinsing magnetic bead of the 200 fresh configurations of μ L, room temperature 30sec is incubated, carefully removes supernatant.
H. it repeats to be rinsed with alcohol primary.
I. keep sample always on magnetic frame, drying at room temperature of uncapping magnetic bead 3min.
J. sample from magnetic frame is taken off, adds in suitable Nuclease free water.Pipettor piping and druming is mixed It is even, it is incubated at room temperature 5min.
K. sample is placed in 2min on magnetic frame, careful to shift in supernatant to new centrifuge tube after solution clarification.
A. library concentration is carried out using Qubit 3.0 to measure and the progress Library Quality assessments of Agilent 2100.
(10) Library Quality assessment 2：Agilent 2100
A. kit (DNA dye concentrate, DNA gel matrix) equilibrium at room temperature 30min.
B. vortex DNA dye concentrate 10sec draw 25 μ L dyestuffs and are added to DNA gel after centrifugation Fully be vortexed mixing in matrix, wink from.
C. it shifts in mixed liquor to spin filter, ± 20% room temperatures of 2240g centrifugation 15min.
D. 4 DEG C of the glue dye centrifuged is kept in dark place.
E. syringe piston is placed at 1.0mL by adjustment glue pressing device cartridge clip in C gears (last step).
F. a new DNA1000 chip is taken out, 9 μ L gel-dye mix are added in the hole labeled as G, and (liquid-transfering gun falls It inhales).
G. chip being placed in priming station and being shut (can hear when priming station are correctly closed " card " sound).
H. injection ram is slowly pressed downward until being blocked by cartridge clip.60sec is kept, discharges cartridge clip, piston rebounds automatically (piston should bounce back into the position of 0.7mL immediately, otherwise possible priming station gas leakage).
I. piston is slowly retracted into 1.0mL positions, opens glue pressing device and take out detection chip.
J. it is marked in the hole having to remaining 2 and adds in 9.0 μ L gel-dye mix (liquid-transfering gun suck-back).
K. in 12 sample wells and enter 5 μ L DNA Marker (green) in 1 Ladder hole.
L. the 1 processed Ladder of μ L are added in Ladder holes, and (Ladder needs to dispense, often 1.1 μ L of pipe, -80 DEG C of guarantors It deposits).
M. 1 μ L samples are added in each sample well, 1 μ L Nuclease free are added in not used hole water。
N. chip mixing is centrifuged into 1min, the chip prepared needs machine testing in 5min, needs to determine before detection every There is no bubble in a hole.
(11) machine on library
Library concentration according to obtained by step (10) is 4.24ng/ μ L, and library mean size is 296bp, and molar concentration is 22.04nM, by library be diluted to it is upper it is confidential ask after, be sequenced using Nextseq500 PE150 strategies, as a result given birth to Object information analysis.
Embodiment 3：A kind of banking process suitable for the analysis of maize leaf transposase accessibility chromatin, with reference to Fig. 1 Flow chart, specific implementation step is as follows：
(1) separation of nucleus.
A. 200mg maize leafs are weighed.
B. tissue is chopped into homogenate shape in culture dish on ice is positioned over sharp blade.
C. 2.5mL TS2 enzymolysis liquids enzymolysis 4h instrument removal cell walls are added in.TS2 enzymolysis liquids are formulated as：1.5% cellulose Enzyme, 0.5% macerozyme, 0.5M mannitol (pH 5.7) mixed liquor is placed in 56 DEG C of water-bath 10min, after being cooled to room temperature, adds in 10mmol/L CaCl2, 0.1%BSA, and with 0.45 μm of membrane filtration.
D. after digesting, 4 DEG C, 400rpm centrifugation 10min discard supernatant liquid.
E. precipitation adds in 2.5mL precooling TS1 lysates, after pipettor blows and beats resuspension repeatedly, is transferred in centrifuge tube and is placed in 100~150rpm vibrates 5min on vortex mixer.The composition of TS1 lysates is：20mM Tris-HCl pH 8.0,75mM KCl, 15mM NaCl, 0.5mM spermine, 10mM beta -mercaptoethanols, 2%PVP, 0.3%TritonX-100.
F. completely after cracking, with magical filter-cloth filtering twice, filtrate is collected.
G. the filtrate layering liquid Percoll gradient centrifugations of density 50%, 47.5%, 42.5%, 37.5%, 400~ 1300rpm centrifuges 25min.
H. the nucleus of separation is collected, 600 μ L TS1 lysates is added to be resuspended.
(2) the DAPI dyeing and sorting of nucleus.
A. nucleus suspension is filtered using 40 μm of sterilizing filter.
B. filtrate is collected to be dyed with DAPI.
C. using flow cytometer, it is sorted according to the size of nucleus.
D. the nucleus centrifugation collected, 4 DEG C, 1300rpm centrifugation 15min are discarded supernatant.
E. precipitation is washed with washing buffer.The composition of washing buffer is：10mM Tris-HCl pH8.0,5mM MgCl2。
(3) Tn5 swivel bases enzyme reaction.
A. swivel base enzyme modification.Transposase is coated with joint sequence：
Adapter1：5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’
3’-TCTACACATATTCTCTGTC-5’
Adapter2：5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’
3’-TCTACACATATTCTCTGTC-5’。
B. the Tn5 transposases and buffer solution added in after modification carries out swivel base reaction.Take 10 μ 5 × TTBL of L Buffer, 5 μ L Tn5 transposases, 50ng DNA, pipettor gently blow and beat of short duration centrifugation after mixing, 37 DEG C of water-bath 50min.
(4) digestion products purify
A. after the completion of swivel base reaction, purified with Qiagen MinElute kit.
B. the Buffer PB (plus pH indicator I) of 5 times of volumes are added in digestion products, are mixed evenly.
C. mixed liquor is transferred to MinElute column, 13000rpm centrifuges 1min at room temperature.
D. filtrate is abandoned, column is put back in 2mL collecting pipes, adds in 750 μ L Buffer PE (plus absolute ethyl alcohol), room The lower 13000rpm centrifugations 1min of temperature.
E. filtrate is abandoned, column is put back in 2mL collecting pipes, 13000rpm skies centrifugation 2min.
F. it shifts in column to clean 1.5mL centrifuge tubes, opens lid, drying at room temperature 3min.
G. column center membranes add in 26 μ L Nuclease free water, be placed at room temperature for 2min, 13000rpm from Heart 2min collects filtrate.
(5) PCR is enriched with
A. prepare following mixed liquor on ice (with embodiment 1)：
B. rear brief centrifugation is mixed evenly, in being reacted as follows on qPCR instrument：
C. each primer sequence is the same as embodiment 1.
(6) quantitative fluorescent PCR determines additional cycles number
A. following mixed liquor is prepared on ice, 5 μ L PCR products is taken to be reacted, resultant product is placed on ice：
B. rear brief centrifugation is mixed evenly, in being reacted as follows on qPCR instrument：
C. the recurring number according to needed for fluorescent quantitative PCR result calculating is additional.Computational methods are right at maximum fluorescence value 1/4 The recurring number answered.According to result of calculation, additional required recurring number is 7cycles, and remaining sample is put into PCR instrument and is continued Reaction.Conversely, it then needs to optimize adjustment to sample initial amount and digestion time.
(7) PCR product purifies
A.PCR products are purified with Qiagen MinElute kit.
The Buffer PB (plus pH indicator I) of 5 times of volumes are added in b.PCR products, are mixed evenly.
C. mixed liquor is transferred to MinElute column, 13000rpm centrifuges 1min at room temperature.
D. filtrate is abandoned, column is put back in 2mL collecting pipes, adds in 750 μ L Buffer PE (plus absolute ethyl alcohol), room The lower 13000rpm centrifugations 1min of temperature.
E. filtrate is abandoned, column is put back in 2mL collecting pipes, 13000rpm skies centrifugation 2min.
F. it shifts in column to clean 1.5mL centrifuge tubes, opens lid, drying at room temperature 3min.
G. 22 μ L Nuclease free water are added in column center membranes, are placed at room temperature for 2min, 13000rpm from Heart 2min collects filtrate.
h.
(8) Library Quality assessment 1：Agilent 2100
A. kit (DNA dye concentrate, DNA gel matrix) equilibrium at room temperature 30min.
B. vortex DNA dye concentrate 10sec draw 25 μ L dyestuffs and are added to DNA gel after centrifugation Fully be vortexed mixing in matrix, wink from.
C. it shifts in mixed liquor to spin filter, ± 20% room temperatures of 2240g centrifugation 15min.
D. 4 DEG C of the glue dye centrifuged is kept in dark place.
E. syringe piston is placed at 1.0mL by adjustment glue pressing device cartridge clip in C gears (last step).
F. a new DNA1000 chip is taken out, 9 μ L gel-dye mix are added in the hole labeled as G, and (liquid-transfering gun falls It inhales).
G. chip being placed in priming station and being shut (can hear when priming station are correctly closed " card " sound).
H. injection ram is slowly pressed downward until being blocked by cartridge clip.60sec is kept, discharges cartridge clip, piston rebounds automatically (piston should bounce back into the position of 0.7mL immediately, otherwise possible priming station gas leakage).
I. piston is slowly retracted into 1.0mL positions, opens glue pressing device and take out detection chip.
J. it is marked in the hole having to remaining 2 and adds in 9.0 μ L gel-dye mix (liquid-transfering gun suck-back).
K. in 12 sample wells and enter 5 μ L DNA Marker (green) in 1 Ladder hole.
L. the 1 processed Ladder of μ L are added in Ladder holes, and (Ladder needs to dispense, often 1.1 μ L of pipe, -80 DEG C of guarantors It deposits).
M. 1 μ L samples are added in each sample well, 1 μ L Nuclease free are added in not used hole water。
N. chip mixing is centrifuged into 1min, the chip prepared needs machine testing in 5min, needs to determine before detection every There is no bubble in a hole.
Data result is as follows：
(9) library fragments screen
A. VAHTS DNA Clean Beads are shifted to an earlier date into 30min from 2-8 DEG C of taking-up, balanced to room temperature, reverse or vortex shakes Swinging makes the abundant mixing of magnetic bead.
B. the magnetic bead of 0.5 × volume (25 μ L), pipettor piping and druming mixing are added according to elution liquor capacity.
C. 5min is incubated at room temperature, DNA is made to be attached on magnetic bead.
D. sample is placed in 5min on magnetic frame, after solution clarification, carefully takes supernatant in new EP pipes.
E. the magnetic bead of 0.7 × volume (35 μ L) of elution liquor capacity, pipettor piping and druming mixing are added in pipe.
F. 5min is incubated at room temperature, sample is placed on magnetic frame, after solution clarification, carefully removes supernatant.
G. sample is kept to be placed on magnetic frame always, adds in the 80% ethyl alcohol rinsing magnetic bead of the 200 fresh configurations of μ L, room temperature 30sec is incubated, carefully removes supernatant.
H. it repeats to be rinsed with alcohol primary.
I. keep sample always on magnetic frame, drying at room temperature of uncapping magnetic bead 3min.
J. sample from magnetic frame is taken off, adds in suitable Nuclease free water.Pipettor piping and druming is mixed It is even, it is incubated at room temperature 5min.
A. sample is placed in 2min on magnetic frame, careful to shift in supernatant to new centrifuge tube after solution clarification.
(10) Library Quality assessment 2：Agilent 2100
A. kit (DNA dye concentrate, DNA gel matrix) equilibrium at room temperature 30min.
B. vortex DNA dye concentrate 10sec draw 25 μ L dyestuffs and are added to DNA gel after centrifugation Fully be vortexed mixing in matrix, wink from.
C. it shifts in mixed liquor to spin filter, ± 20% room temperatures of 2240g centrifugation 15min.
D. 4 DEG C of the glue dye centrifuged is kept in dark place.
E. syringe piston is placed at 1.0mL by adjustment glue pressing device cartridge clip in C gears (last step).
F. a new DNA1000 chip is taken out, 9 μ L gel-dye mix are added in the hole labeled as G, and (liquid-transfering gun falls It inhales).
G. chip being placed in priming station and being shut (can hear when priming station are correctly closed " card " sound).
H. injection ram is slowly pressed downward until being blocked by cartridge clip.60sec is kept, discharges cartridge clip, piston rebounds automatically (piston should bounce back into the position of 0.7mL immediately, otherwise possible priming station gas leakage).
I. piston is slowly retracted into 1.0mL positions, opens glue pressing device and take out detection chip.
J. it is marked in the hole having to remaining 2 and adds in 9.0 μ L gel-dye mix (liquid-transfering gun suck-back).
K. in 12 sample wells and enter 5 μ L DNA Marker (green) in 1 Ladder hole.
L. the 1 processed Ladder of μ L are added in Ladder holes, and (Ladder needs to dispense, often 1.1 μ L of pipe, -80 DEG C of guarantors It deposits).
M. 1 μ L samples are added in each sample well, 1 μ L Nuclease free are added in not used hole water。
N. chip mixing is centrifuged into 1min, the chip prepared needs machine testing in 5min, needs to determine before detection every There is no bubble in a hole.
(11) machine on library
Library concentration according to obtained by step (10) is 5.06ng/ μ L, and library mean size is 320bp, and molar concentration is 24.33nM, by library be diluted to it is upper it is confidential ask after, be sequenced using Nextseq500 PE150 strategies, as a result given birth to Object information analysis.
Embodiment described above is a kind of preferable scheme of the present invention, and not the present invention is made in any form Limitation also has other variants and remodeling under the premise of without departing from the technical solution recorded in claim.
Sequence table
<110>It is difficult to understand bright（Hangzhou）Gene Tech. Company Limited
<120>A kind of banking process suitable for the analysis of plant transposase accessibility chromatin
<130> 2017.12
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<170> SIPOSequenceListing 1.0
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tcgtcggcag cgtcagatgt gtataagaga cag 33
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence ()
<400> 2
tctacacata ttctctgtc 19
<210> 3
<211> 34
<212> DNA
<213>Artificial sequence ()
<400> 3
gtctcgtggg ctcggagatg tgtataagag acag 34
<210> 4
<211> 19
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<400> 4
tctacacata ttctctgtc 19
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<213>Artificial sequence ()
<400> 5
aatgatacgg cgaccaccga gatctacact cgtcggcagc gtcagatgt 49
<210> 6
<211> 44
<212> DNA
<213>Artificial sequence ()
<400> 6
caagcagaag acggcatacg agatgtctcg tgggctcgga gatg 44
<210> 7
<211> 29
<212> DNA
<213>Artificial sequence ()
<400> 7
aatgatacgg cgaccaccga gatctacac 29
<210> 8
<211> 24
<212> DNA
<213>Artificial sequence ()
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caagcagaag acggcatacg agat 24

Claims (10)

1. a kind of banking process suitable for the analysis of plant transposase accessibility chromatin, which is characterized in that including following step Suddenly：
S1：Plant tissue sample obtains nuclei suspension A after enzymolysis liquid TS2 and lysate TS1 processing；
S2：Nucleus in nuclei suspension A is dyed, is sorted, obtains nucleus B；
S3：Swivel base reaction and purifying are carried out to nucleus B, obtains swivel base product C；
S4：PCR amplification is carried out to swivel base product C, obtains amplified production D；
S5：Part amplified production D is taken to carry out qPCR with determine it is additional needed for PCR cycle number, according to it is determining it is additional needed for PCR follow Number of rings continues to expand to remaining amplified production D, obtains amplified production E；
S6：Purifying amplified production E obtains single library, and single library carries out segment sorting, obtains machine library.
2. banking process according to claim 1, which is characterized in that S1 is as follows：Take 200~300mg plants Tissue is chopped into homogenate shape in culture dish on ice is positioned over blade, adds in 2.5~3.0mL enzymolysis liquid TS2 enzymes by tissue Solve 4h removal cell walls；4 DEG C, 300~400rpm centrifugation 10min after abandon supernatant；Add in the lysate of 2.5~3.0mL precoolings TS1 is transferred in centrifuge tube and is placed in 100~150rpm oscillations 5min on vortex mixer, obtains the mixed liquor after cracking completely；It will be mixed Close liquid filtering twice, gained filtrate is distributed in the layering liquid of different densities, and 1000~1300rpm centrifuges 20~25min, is collected The nucleus of separation adds in 600 μ L lysate TS1, obtains nuclei suspension A.
3. banking process according to claim 1, which is characterized in that S2 is as follows：Nuclei suspension A is made Filtered with 40 μm of sterilizing filters, collect filtrate and carry out nuclear targeting and simultaneously sorted according to nucleus size, sorting it is thin Karyon is collected using the centrifuge tube for being previously charged into 600 μ L lysates TS1；The cell caryoplasm that micro- Microscopic observation sorts After amount, 4 DEG C of nucleus being collected into, 1000~1300rpm are centrifuged into 15min, abandon supernatant, 600 μ L washing buffers is added in and washes Nucleus is washed, abandoning supernatant after 1000~1300rpm centrifugations 10min obtains nucleus B.
4. banking process according to claim 1, which is characterized in that the washing buffer component forms as follows：10mM、 The Tris-HCl of pH8.0,5mM MgCl2。
5. banking process according to claim 1, which is characterized in that S3 is as follows：Nucleus B is taken to carry out immediately Swivel base reacts, after nucleus B adds in Tn5 transposases, 37 DEG C of 30~70min of water-bath；Swivel base product after water-bath is carried out Purifying, obtains swivel base product C.
6. banking process according to claim 1 or 2, which is characterized in that the enzymolysis liquid TS2 components form as follows： 0.5%~1.5% cellulase, 0.3%~0.6% pectase, 0.3%~0.6% hemicellulase, 0.45~0.55M are sweet Reveal alcohol, 2- (N- morpholines) ethanesulfonic acid monohydrate of 20mM KCl, 10mM, pH5.7,10mM CaCl2, 0.1% bovine serum albumin In vain.
7. according to the banking process described in claims 1 or 2 or 3, which is characterized in that the lysate TS1 components form as follows： 20mM Tris-HCl pH 8.0,75mM KCl, 15mM NaCl, 0.5mM spermine, 10mM beta -mercaptoethanols, 2%PVP, 0.3% TritonX-100。
8. banking process according to claim 1, which is characterized in that additional required PCR cycle number is determined in S5, is calculated Mode is as follows：According to quantitative fluorescent PCR curve graph, corresponding recurring number is additional required cycle when taking maximum fluorescence value 1/4 Number.
9. banking process according to claim 1, which is characterized in that S6 is as follows：Amplified production E is distinguished pure After change, single library is obtained, measures the concentration in single library, is mixed into multiple single library equimolar concentrations according to concentration mixed Close library, mixing library using two-wheeled DNA magnetic beads carry out segment sorting, retain 200~1000bp DNA fragmentation obtain sorting after Library carries out Library Quality detection using foranalysis of nucleic acids instrument, machine library is obtained after detection is qualified.
10. banking process according to claim 9, which is characterized in that mixing library carries out segment using two-wheeled DNA magnetic beads Sorting, the volume ratio of first round magnetic bead for 0.4 ×~0.9 ×, the volume ratio of the second wheel magnetic bead for 0.5 ×~1.0 ×.
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CN113444678A (en) * 2020-03-26 2021-09-28 武汉华大医学检验所有限公司 Method for preparing single cell nuclear suspension, single cell sequencing method and kit
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