CN115044646A - Direct RNA sequencing and database building method - Google Patents
Direct RNA sequencing and database building method Download PDFInfo
- Publication number
- CN115044646A CN115044646A CN202210250375.8A CN202210250375A CN115044646A CN 115044646 A CN115044646 A CN 115044646A CN 202210250375 A CN202210250375 A CN 202210250375A CN 115044646 A CN115044646 A CN 115044646A
- Authority
- CN
- China
- Prior art keywords
- rna
- tube
- room temperature
- buffer
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a direct RNA sequencing and database building method, which comprises PolyA + The invention relates to a method for realizing RNA enrichment, an A adding reaction without polyA tail RNA and library establishment.
Description
Technical Field
The invention relates to the technical field of gene sequencing, in particular to a direct RNA sequencing and database building method.
Background
The conventional RNA direct sequencing based on the ONT sequencing platform adopts an SQK-RNA002 library building kit to complete library building according to the operation flow shown in figure 1.
The method comprises the following specific steps:
RNA dosage: the total volume is 9 mu L and the total volume is 500 ng;
2. preparing a PCR Reaction system comprising NEBNext Quick Ligation Reaction Buffer 3.0 mu L, RNA 9.0.0 mu L, RNA CS (RCS)110nM 0.5 mu L, RT Adapter (RTA)1.0 mu L, T4 DNA ligase 1.5 mu L, mixing all the components, quickly centrifuging, and incubating at room temperature for 10 min;
3. preparing a reverse transcription reaction system comprising: nuclease-free water 9.0. mu.L, 10mM dNTPs 2.0. mu.L, 5x first-strand buffer 8.0. mu.L, 0.1M DTT 4.0. mu.L;
4. adding the mixed solution obtained in the step 3 into the reaction system obtained in the step 2, and uniformly mixing;
5. adding 2 mu L of Super Script III reverse transcriptase to the reaction system, and mixing uniformly;
6. setting a PCR amplification program, storing at 50 ℃ for 50 minutes, at 70 ℃ for 10 minutes and at 4 ℃ respectively, and carrying out PCR amplification;
7. transferring the PCR product in the step 6 into a 1.5mL sterile centrifuge tube;
8. vortex and shake and resuspend Agencour RNA Clean XP beads, take 72 μ L and add to the solution of step 6, incubate 5 minutes in Hula mixer at room temperature after pipettor mixing evenly;
9. placing the sample tube on a magnetic frame, standing, sucking out supernatant, washing magnetic beads with 150 μ L of 70% ethanol, and sucking out 70% ethanol with a pipette;
10. taking the centrifuge tube off the magnetic frame, resuspending the magnetic beads with 20. mu.L of nuclease-free water, and incubating for 5 minutes at room temperature;
11. transferring the eluent into a 1.5ml sterile centrifuge tube, preparing an Adapter connection Reaction system, comprising 20.0 mu L of RNA solution, 8.0 mu L of NEB Next Quick Ligation Reaction Buffer, 6.0 mu L of RNA Adapter (RMX), 3.0 mu L, T4 DNA Ligase 3.0 mu L, T4 DNA Ligase 3.0 mu L of nuclease-free water, mixing the components uniformly, and incubating for 10 minutes at room temperature;
12. adding 40 mu L of RNA Clean XP beads into the joint connection reaction system in the step 11, uniformly mixing, and then incubating for 5 minutes in a Hula mixer at room temperature;
13. the sample tube was placed on a magnetic stand, the supernatant was aspirated off, 150. mu.L of wash buffer (WSB) was added to wash the magnetic beads, and the supernatant was removed. Cleaning is repeated for one time;
14. the beads were resuspended by adding 21. mu.L of Elution Buffer (EB) and incubated for 10 minutes at room temperature. And (5) sucking the supernatant into a 1.5mL sterile centrifuge tube, and taking 1 mu L of the supernatant for nucleic acid concentration detection to complete library establishment.
The above library building method has the following defects: the method can only be used for constructing a library of RNA containing poly A tail; each sequencing chip can only sequence one library.
Disclosure of Invention
The invention provides a direct RNA sequencing and database building method.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: a direct RNA sequencing database construction method comprising the steps of:
S1、Poly A + enrichment of RNA comprising:
s1.1, annealing of probe
a. Adding 0.1-1.0mg total RNA (RNA equal to or more than 50 μ g), placing in 1.5mL RNase-free sterile centrifuge tube, adding RNase-free water to make up volume to 500 μ L,
b. the centrifuge tubes were incubated at 65 ℃ for 10 minutes,
c. add 3. mu.L of biotinylated oligo (dT) probe, 13. mu.L of 20 XSSC to the RNA solution, mix gently, and cool at room temperature;
s1.2, preparing a reaction buffer solution, and preparing a 0.5 XSSC buffer solution and a 0.1 XSSC buffer solution in the process of waiting for the RNA solution to be cooled (the cooling time is more than or equal to 10 minutes), wherein the method specifically comprises the following steps:
d. 1.2mL of sterile 0.5 XSSC buffer was prepared, 30. mu.L of 20 XSSC buffer was added with 1.170mL of RNase-Free water and mixed well,
b. prepare 1.4mL of sterile 0.1 XSSC buffer, including: 7 mu.L of 20 XSSC buffer solution is added with 1.393mL of RNase-Free water and mixed evenly;
s1.3, washing magnetic beads, comprising:
e. resuspending a tube of streptavidin magnetic beads (SA-PMPs)0.6mL, flicking the tube bottom until the magnetic beads are fully dispersed, placing the tube on a magnetic frame to make the magnetic beads be adsorbed to one side of the tube wall,
f. the supernatant was carefully removed, without centrifugation,
g. the SA-PMPs were washed three times with 300. mu.L of 0.5 XSSC buffer, magnetic beads were collected by magnetic adsorption on a magnetic rack each time, the supernatant was carefully removed,
h. finally resuspend SA-PMPs with 100. mu.L of 0.5 XSSC;
s1.4, capturing and washing oligo (dT) -mRNA hybrid strand, comprising:
i. adding all reaction products obtained in the step S1.1 and in the probe annealing process into the cleaned SA-PMPs, gently mixing the reaction products,
j. incubating for 10 minutes at room temperature, slightly inverting and mixing the mixture once every 1 to 2 minutes,
k. adsorbing and collecting SA-PMPs by a magnetic frame, carefully sucking the solution into a sterile 1.5mL centrifuge tube without touching magnetic beads, placing the centrifuge tube on ice for later use,
m, washing the magnetic beads by using 300 mu L of 0.1 XSSC buffer solution, flicking the bottom of the tube until the magnetic beads are fully resuspended, removing supernatant, and repeatedly washing for four times;
s1.5, elution of mRNA, including:
n, resuspending the SA-PMPs in step S1.4 with 100. mu.L of RNase-free water, resuspending the magnetic beads at the bottom of the flick tube,
o, adsorbing the magnetic beads by a magnetic frame, absorbing the eluted mRNA solution into a sterile centrifuge tube, simultaneously retaining the magnetic beads,
p, washing SA-PMPs with 150. mu.L of RNase-free water repeatedly once, sucking the solution into the tube in the previous step,
s1.6, purification, concentration of eluted mRNA (RNA Clean)&Concentrator TM -5)
q, buffer preparation: adding 48mL of absolute ethanol to 12mL of RNA Wash buffer,
r, adding RNA binding buffer with twice volume to each sample tube in the step S1.5 for eluting mRNA, mixing uniformly, adding absolute ethyl alcohol with the same volume, mixing uniformly, adding the mixed solution to Zymo-Spin put in a collecting tube TM In IICR Column, centrifuging for 1 minute at room temperature, discarding the liquid in the collection tube,
s, adding 400 mu L of RNA prep buffer into an adsorption column, centrifuging for 1 minute at the room temperature of 12000g, discarding the liquid in a collection tube,
t, adding 700 mu L of RNA wash buffer into an adsorption column, centrifuging for 1 minute at the room temperature of 12000g, discarding the liquid in a collecting pipe,
u, adding 400 mu L of RNA wash buffer into the adsorption column, centrifuging for 1 minute at the room temperature of 12000g, discarding the liquid in the collection tube, centrifuging the empty tube for 1 minute at the room temperature again, completely removing the wash buffer, carefully moving the column to an RNase-free centrifuge tube,
v, adding 10 mu L of RNase-free water into the adsorption column, centrifuging for 1 minute at room temperature to elute RNA,
s1.7, taking 1 mu L of the RNA solution in the step S1.6, and detecting the nucleic acid concentration by using a Qubit 3.0;
s2, addition of A reaction without poly A tail RNA
The solution of step S1.4 contains RNA, rRNA and tRNA without poly A, and RNA Clean is used&Concentrator TM -25, carrying out the purification by using the kit,
s2.1, adding RNA binding buffer with twice volume to each sample tube, adding absolute ethyl alcohol with the same volume after mixing uniformly, adding the mixed solution to Zymo-Spin after mixing uniformly TM In IICR Column, centrifuging for 1 minute at room temperature, discarding the liquid in the collection tube,
1) adding 400 mu L of RNA prep buffer into the adsorption column, centrifuging for 1 minute at room temperature, discarding the liquid in the collection tube,
2) adding 700 mu L of RNA wash buffer into an adsorption column, centrifuging for 1 minute at room temperature, discarding the liquid in a collection tube,
3) adding 400 mu L of RNA wash buffer into an adsorption column, centrifuging for 1 minute at room temperature, discarding the liquid in a collection tube,
4) the empty tube was centrifuged again for 1 minute at room temperature to completely remove the wash buffer. Carefully move the column to the RNase-free centrifuge tube,
5) adding 30 μ L RNase-free water into the adsorption column, centrifuging at room temperature for 1 min to elute RNA,
6) preparing an A adding reaction system as follows:
30. mu.L of the RNA solution in step 5), 10 XE. coli Poly (A) Polymerase Reaction Buffer 4. mu.L (1X), 4. mu.L of ATP (10mM), 2. mu.L of E.coli Poly (A) Polymerase in a total volume of 40. mu.L;
7) the reaction solution was incubated at 37 ℃ for 30 minutes, and then EDTA was added thereto to a final concentration of 10mM, to terminate the reaction,
8) by RNA Clean&Concentrator TM -5 kit purification of the reaction product of step 7), in particular by:
a1, adding two volumes of RNA binding buffer to each sample tube, and mixing. Adding equal volume of absolute ethyl alcohol, mixing evenly,
a2, adding the mixture to Zymo-Spin TM In IICR Column, 12000g of the solution is centrifuged for 1 minute at room temperature, the liquid in a collecting tube is discarded,
a3, adding 400 mu L of RNA prep buffer into an adsorption column, centrifuging for 1 minute at 12000g, discarding the liquid in a collection tube,
a4, adding 700 mu L of RNA wash buffer into the adsorption column, centrifuging for 1 minute at 12,000g, discarding the liquid in the collection tube,
a5, adding 400 mu L RNA wash buffer into the adsorption column, centrifuging for 1 minute at 12000g, discarding the liquid in the collection tube,
a6, centrifuging the empty tube again, centrifuging the tube at room temperature for 1 minute, completely removing the wash buffer,
a7, carefully transferring the adsorption column to the RNase-free centrifuge tube, adding 10. mu.L of RNase-free water to the column, centrifuging at 12,000g for 1 minute, eluting RNA,
9) taking 1 mu L of the eluted RNA solution, and detecting the concentration of nucleic acid by using a Qubit 3.0;
s3, establishing a library, comprising:
b1, RNA dosage: the total volume is 9. mu.L, the total volume is 500ng, namely the RNA products obtained in the step S1 and the step S2 are respectively subjected to library construction,
b2, preparing the following reaction system in a PCR tube:
NEBNext Quick Ligation Reaction Buffer 3.0 μ L, RNA 9.0.0 μ L, RNA CS (RCS)110nM 0.5 μ L, Barcode 1/Barcode 2/Barcode 3/Barcode 4/RTAdapter (RTA)1.0 μ L, T4 DNA strain 1.5 μ L, total volume 15 μ L, components of these volumes were mixed and then centrifuged rapidly, incubated at room temperature for 10 minutes, 4 pairs of Barcode of HPLC grade were synthesized based on the nucleotide sequence information of Barcode 1/Barcode 2/Barcode 3/Barcode 4, A and B components of each Barcode were diluted with RNase-free water to a final concentration of 10 μ M, respectively, and A and B components of each pair of Barcode were mixed in equal volumes before use;
b3, preparing a reaction system for reverse transcription, which comprises 9.0. mu.L of nuclease-free water, 2.0. mu.L of 10mM dNTPs, 8.0. mu.L of 5 Xfirst-strand buffer, 4.0. mu.L of 0.1M DTT, and a total volume of 23.0. mu.L,
b4, adding the mixed solution in the step b3 into the reaction system in the step b2, mixing evenly,
b5, adding 2 mu L SuperScript III reverse transcriptase into the reaction system, mixing evenly,
b6, setting a PCR amplification program, keeping at 50 ℃ for 50 minutes, 70 ℃ for 10 minutes and 4 ℃ respectively, carrying out PCR amplification,
b7, transferring the PCR product in the step b6 into a sterile 1.5mL centrifuge tube,
b8, resuspending Agencour RNAclean XP beads by vortex shaking, sucking 72 mu L of Agencour RNAclean XP beads into the reaction system in the step b7, uniformly mixing by a pipette, and incubating for 5 minutes at room temperature in a Hula mixer,
b9, placing the sample tube on a magnetic frame, operating on the magnetic frame, sucking the supernatant, washing the magnetic beads with 150 mu L of 70% ethanol, finally sucking 70% ethanol by a pipette,
b10, removing the sample tube from the magnetic frame, resuspending the magnetic beads in 20. mu.L of nuclease-free water, incubating at room temperature for 5 minutes,
b11, adding the 20. mu.L of eluent into a sterile 1.5mL Eppendorf DNA Lobind tube, preparing a joint connection reaction system: comprises 20.0 mu L of RNA solution in the step b10, 8.0 mu L of NEBNext Quick Ligation Reaction Buffer, 6.0 mu L of RNA Adapter (RMX) and 3.0 mu L of nuclease-free water, the components are mixed evenly and incubated for 10 minutes at room temperature,
b12, adding 40 mu L of RNAclean XP beads into the joint connection reaction system, evenly mixing, incubating for 5 minutes in a Hula mixer at room temperature,
b13, placing the sample tube on a magnetic frame, sucking the supernatant, adding 150 mu L of wash buffer (WSB) to clean the magnetic beads, sucking the supernatant, repeating the cleaning once,
b14, adding 21 mu L of elution buffer solution (EB), resuspending magnetic beads, incubating at room temperature for 10 minutes, placing the tube on a magnetic frame, sucking supernatant into a sterile centrifuge tube, taking 1 mu L of the supernatant, and detecting the nucleic acid concentration to complete library establishment.
As an improvement, the cooling time of the RNA solution in the step S1.2 is more than or equal to 10 minutes.
As an improvement, the solution for capturing and washing oligo (dT) -mRNA hybrid strand in step S1.4 contains RNA without ploy A tail.
As a modification, the step a3, the step a4 and the step a5 are all centrifuged at room temperature.
The invention has the following advantages: the library construction scheme is suitable for a Nanopore gene sequencer, and can be used for direct RNA sequencing of a plurality of samples on one chip by matching with a sequencing chip and a library construction kit. In the direct RNA sequencing library construction method provided by the Nanopore official, only one chip can be used for detecting one library, and only RNA with poly A tail can be used for constructing the library. The successful addition of A and library construction sequencing of poly A-free RNA increases the utilization rate of the sample, and is more beneficial to the use of the precious sample with low sample concentration. In general, the technology of the invention increases RNA sequencing data quantity, improves sequencing flux and reduces library construction cost.
Drawings
FIG. 1 is a schematic diagram of SQK-RNA002 library construction flow of the direct RNA sequencing library construction method of the present invention.
FIG. 2 is a schematic diagram of the experimental procedure of a direct RNA sequencing and database construction method of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
With reference to fig. 2, a direct RNA sequencing database construction method comprises the following steps:
S1、Poly A + enrichment of RNA comprising:
s1.1, annealing of probe
a. Adding 0.1-1.0mg total RNA (RNA equal to or more than 50 μ g), adding into 1.5mL sterile centrifuge tube of RNase-free, adding RNase-free water to make up the volume to 500 μ L,
b. the centrifuge tubes were incubated at 65 ℃ for 10 minutes,
c. adding 3. mu.L of Biotinylated-oligo (dT) probe and 13. mu.L of 20 XSSC to the RNA solution, gently mixing, and standing at room temperature until the mixture is cooled;
s1.2, preparing a reaction buffer solution, and preparing a 0.5 XSSC and a 0.1 XSSC buffer solution in the process of waiting for the cooling of the RNA solution (the cooling time is more than or equal to 10 minutes), wherein the method specifically comprises the following steps:
d. 1.2mL of sterile 0.5 XSSC buffer was prepared, 30. mu.L of 20 XSSC buffer was added with 1.170mL of RNase-Free water and mixed well,
b. prepare 1.4mL of sterile 0.1 XSSC buffer, including: 7 mu.L of 20 XSSC buffer solution is added with 1.393mL of RNase-Free water and mixed evenly;
s1.3, washing magnetic beads, comprising:
e. resuspending a tube of streptavidin magnetic beads (SA-PMPs)0.6mL, flicking the tube bottom until the magnetic beads are fully dispersed, placing the tube on a magnetic frame to make the magnetic beads be adsorbed to one side of the tube wall,
f. the supernatant was carefully removed, without centrifugation,
g. the SA-PMPs were washed three times with 300. mu.L of 0.5 XSSC buffer, magnetic beads were collected by magnetic adsorption on a magnetic rack each time, the supernatant was carefully removed,
h. finally resuspend SA-PMPs with 100. mu.L of 0.5 XSSC;
s1.4, capturing and washing oligo (dT) -mRNA hybrid strand, comprising:
i. adding all reaction products obtained in the step S1.1 and in the probe annealing process into the cleaned SA-PMPs, gently mixing the reaction products,
j. incubating for 10 minutes at room temperature, slightly inverting and mixing the mixture once every 1 to 2 minutes,
k. adsorbing and collecting SA-PMPs by a magnetic frame, carefully sucking the solution into a sterile 1.5mL centrifuge tube without touching magnetic beads, placing the centrifuge tube on ice for later use,
m, washing the magnetic beads by using 300 mu L of 0.1 XSSC buffer solution, flicking the tube bottom until the magnetic beads are fully resuspended, removing supernatant, and repeatedly washing for four times;
s1.5, elution of mRNA, including:
n, resuspending the SA-PMPs in step S1.4 with 100. mu.L of RNase-free water, resuspending the magnetic beads at the bottom of the flick tube,
o, adsorbing the magnetic beads by a magnetic frame, absorbing the eluted mRNA solution into a sterile centrifuge tube, simultaneously retaining the magnetic beads,
p, washing SA-PMPs with 150. mu.L of RNase-free water repeatedly once, sucking the solution into the tube in the previous step,
s1.6, purification, concentration of eluted mRNA (RNA Clean)&Concentrator TM -5)
q, preparation of buffer: adding 48mL of absolute ethanol to 12mL of RNA Wash buffer,
r, adding RNA binding buffer with twice volume to each sample tube in the step S1.5 for eluting mRNA, mixing uniformly, adding absolute ethyl alcohol with the same volume, mixing uniformly, adding the mixed solution to Zymo-Spin put in a collecting tube TM In IICR Column, centrifuging for 1 minute at room temperature, discarding the liquid in the collection tube,
s, adding 400 mu L of RNA prep buffer into an adsorption column, centrifuging for 1 minute at the room temperature of 12000g, discarding the liquid in a collection tube,
t, adding 700 mu L of RNA wash buffer into the adsorption column, centrifuging for 1 minute at 12000g at room temperature, discarding the liquid in the collection tube,
u, adding 400 mu L of RNA wash buffer into the adsorption column, centrifuging for 1 minute at the room temperature of 12000g, discarding the liquid in the collection tube, centrifuging the empty tube again at the room temperature of 12000g for 1 minute, completely removing the wash buffer, carefully moving the column to an RNase-free centrifuge tube,
v, adding 10 mu L of RNase-free water into the adsorption column, centrifuging at the room temperature of 12000g for 1 minute to elute RNA,
s1.7, taking 1 mu L of the RNA solution in the step S1.6, and detecting the nucleic acid concentration by using a Qubit 3.0;
s2, addition of A reaction without poly A tail RNA
The solution of step S1.4 contains RNA without poly A tail, rRNA and tRNA, and RNA Clean is used&Concentrator TM -25, carrying out the purification by using the kit,
s2.1, adding RNA binding buffer with twice volume to each sample tube, adding absolute ethyl alcohol with the same volume after mixing uniformly, adding the mixed solution to Zymo-Spin after mixing uniformly TM In IICR Column, centrifuging for 1 minute at room temperature, discarding the liquid in the collection tube,
1) adding 400 mu L of RNA prep buffer into an adsorption column, centrifuging for 1 minute at room temperature, discarding the liquid in a collection tube,
2) adding 700 mu L of RNA wash buffer into an adsorption column, centrifuging for 1 minute at room temperature, discarding the liquid in a collection tube,
3) adding 400 mu L of RNA wash buffer into an adsorption column, centrifuging for 1 minute at room temperature, discarding the liquid in a collection tube,
4) the empty tube was centrifuged again for 1 minute at room temperature to completely remove the wash buffer. Carefully move the column to the RNase-free centrifuge tube,
5) adding 30 μ L RNase-free water into the adsorption column, centrifuging at room temperature for 1 min to elute RNA,
6) preparing an A adding reaction system as follows:
30. mu.L of the RNA solution in step 5), 10 XE. coli Poly (A) Polymerase Reaction buffer 4. mu.L (1X), 4. mu.L of ATP (10mM), 2. mu.L of E.coli Poly (A) Polymerase in a total volume of 40. mu.L;
7) the reaction solution was incubated at 37 ℃ for 30 minutes, and then EDTA was added thereto to a final concentration of 10mM, to terminate the reaction,
8) by RNA Clean&Concentrator TM -5 kit purification of the reaction product of step 7), in particular by:
a1, adding two volumes of RNA binding buffer to each sample tube, and mixing. Adding equal volume of absolute ethyl alcohol, mixing evenly,
a2, adding the mixture to Zymo-Spin TM In IICR Column, 12000g of the solution is centrifuged for 1 minute at room temperature, the liquid in a collecting tube is discarded,
a3, adding 400 mu L of RNA prep buffer into an adsorption column, centrifuging for 1 minute at 12000g, discarding the liquid in a collection tube,
a4, adding 700 mu L RNA wash buffer into the adsorption column, centrifuging for 1 minute at 12000g, discarding the liquid in the collection tube,
a5, adding 400 mu L RNA wash buffer into the adsorption column, centrifuging for 1 minute at 12000g, discarding the liquid in the collection tube,
a6, centrifuging the empty tube again at room temperature for 1 minute to completely remove the wash buffer,
a7, carefully transferring the adsorption column to the RNase-free centrifuge tube, adding 10. mu.L RNase-free water to the column, centrifuging at 12000g for 1 min, eluting RNA,
9) taking 1 mu L of the eluted RNA solution, and detecting the concentration of nucleic acid by using a Qubit 3.0;
s3, establishing a library, comprising:
b1, RNA dosage: the total volume is 9. mu.L, the total volume is 500ng, namely the RNA products obtained in the step S1 and the step S2 are respectively subjected to library construction,
b2, preparing the following reaction system in a PCR tube:
NEBNext Quick Ligation Reaction Buffer 3.0 μ L, RNA 9.0.0 μ L, RNA CS (RCS)110nM 0.5 μ L, Barcode 1/Barcode 2/Barcode 3/Barcode 4/RTAdap (RTA)1.0 μ L, T4 DNA strain 1.5 μ L, total volume 15 μ L, components of these volumes were mixed and then centrifuged rapidly, incubated at room temperature for 10 minutes, 4 pairs of Barcode of HPLC grade were synthesized based on nucleotide sequence information of Barcode 1/Barcode 2/Barcode 3/Barcode 4, A and B components of each pair of Barcode were diluted with RNase-free water to final concentrations of 10 μ M, respectively, and A and B components of each pair of Barcode were mixed in equal volumes before use;
b3, preparing a reaction system for reverse transcription, which comprises 9.0. mu.L of nuclease-free water, 2.0. mu.L of 10mM dNTPs, 8.0. mu.L of 5 Xfirst-strand buffer, 4.0. mu.L of 0.1M DTT, and a total volume of 23.0. mu.L,
b4, adding the mixed solution in the step b3 into the reaction system in the step b2, uniformly mixing,
b5, adding 2 mu L SuperScript III reverse transcriptase into the reaction system, mixing evenly,
b6, setting a PCR amplification program, keeping at 50 ℃ for 50 minutes, 70 ℃ for 10 minutes and 4 ℃ respectively, carrying out PCR amplification,
b7, transferring the PCR product in the step b6 into a sterile 1.5mL centrifuge tube,
b8, resuspending Agencourt RNAclean XP beads by vortex shaking, sucking 72 mu L of Agencourt RNAclean XP beads to the reaction system in the step b7, uniformly mixing by using a pipette, and incubating for 5 minutes at room temperature in a Hula mixer,
b9, placing the sample tube on a magnetic frame, operating on the magnetic frame, sucking the supernatant, washing the magnetic beads with 150 mu L of 70% ethanol, finally sucking 70% ethanol by a pipette,
b10, removing the sample tube from the magnetic frame, resuspending the magnetic beads in 20. mu.L of nuclease-free water, incubating at room temperature for 5 minutes,
b11, adding the 20 mu L of eluent into a sterile 1.5mL Eppendorf DNA Lobind tube, and preparing a joint connection reaction system: comprises 20.0 mu L of RNA solution in the step b10, 8.0 mu L of NEBNext Quick Ligation Reaction Buffer, 6.0 mu L of RNA Adapter (RMX) and 3.0 mu L of nuclease-free water, the components are mixed evenly and incubated for 10 minutes at room temperature,
b12, adding 40 mu L of RNAclean XP beads into the joint connection reaction system, evenly mixing, incubating for 5 minutes in a Hula mixer at room temperature,
b13, placing the sample tube on a magnetic frame, sucking the supernatant, adding 150 mu L of wash buffer (WSB) to clean the magnetic beads, sucking the supernatant, repeating the cleaning once,
b14, adding 21 mu L of elution buffer solution (EB), resuspending magnetic beads, incubating at room temperature for 10 minutes, placing the tube on a magnetic frame, sucking supernatant into a sterile centrifuge tube, taking 1 mu L of the supernatant, and detecting the nucleic acid concentration to complete library establishment.
The solution for capturing and washing oligo (dT) -mRNA hybrid strands in step S1.4 contains RNA without a ploy A tail.
In the specific implementation of the invention, the mitochondrial RNA extracted from the human 293 cell is used for testing the method; carrying out the first experiment by adopting a conventional library building method; experiment two is carried out by adopting the library building method; the sequencing results of two database building methods were compared based on the ONT sequencing platform as follows:
the results show that the database construction method of experiment two produces twice the amount of data as that of experiment one, and that the value of N50 is larger, resulting in longer sequence length.
The working principle of the invention is as follows: the nucleotide sequence information of Barcode 1/Barcode 2/Barcode 3/Barcode 4 is shown in the following table:
the present invention and its embodiments have been described above, but the description is not limitative, and the actual structure is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Beijing Heijieshenwei Biotech Co., Ltd
<120> direct RNA sequencing and library building method
<130> 2022-07-26
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 38
<212> DNA
<213> Artificial sequence
<400> 1
cctcccctaa aaacgagccg catttgcgta gtaggttc 38
<210> 2
<211> 57
<212> DNA
<213> Artificial sequence
<400> 2
gaggcgagcg gtcaattttc gcaaatgcgg ctcgttttta ggggaggttt ttttttt 57
<210> 3
<211> 38
<212> DNA
<213> Artificial sequence
<400> 3
cctcgtcggt tctaggcatc gcgtatgcta gtaggttc 38
<210> 4
<211> 57
<212> DNA
<213> Artificial sequence
<400> 4
gaggcgagcg gtcaattttg catacgcgat gcctagaacc gacgaggttt ttttttt 57
<210> 5
<211> 38
<212> DNA
<213> Artificial sequence
<400> 5
cctcccactt tcacacgcac taaccaggta gtaggttc 38
<210> 6
<211> 57
<212> DNA
<213> Artificial sequence
<400> 6
gaggcgagcg gtcaattttc ctggttagtg cgtgtgaaag tgggaggttt ttttttt 57
<210> 7
<211> 38
<212> DNA
<213> Artificial sequence
<400> 7
cctccttcag aagagggtcg cttctaccta gtaggttc 38
<210> 8
<211> 57
<212> DNA
<213> Artificial sequence
<400> 8
gaggcgagcg gtcaattttg gtagaagcga ccctcttctg aaggaggttt ttttttt 57
Claims (5)
1. A direct RNA sequencing and library building method is characterized in that: the method comprises the following steps:
S1、Poly A + enrichment of RNA comprising:
s1.1, annealing of probe
a. Adding 0.1-1.0mg total RNA, placing in 1.5mL RNase-free sterile centrifuge tube, adding RNase-free water to make up the volume to 500 μ L,
b. the centrifuge tubes were incubated at 65 ℃ for 10 minutes,
c. add 3. mu.L of biotinylated oligo (dT) probe, 13. mu.L of 20 XSSC to the RNA solution, mix gently, and cool at room temperature;
s1.2, preparing a reaction buffer solution, and preparing a 0.5 XSSC and a 0.1 XSSC buffer solution in the process of waiting for the RNA solution to be cooled, wherein the method specifically comprises the following steps:
d. 1.2mL of sterile 0.5 XSSC buffer was prepared, 30. mu.L of 20 XSSC buffer was added with 1.170mL of RNase-Free water and mixed well,
b. prepare 1.4mL of sterile 0.1 XSSC buffer, including: 7 mu.L of 20 XSSC buffer solution is added with 1.393mL of RNase-Free water and mixed evenly;
s1.3, washing magnetic beads, comprising:
e. resuspending a tube of streptavidin magnetic beads with 0.6mL, flicking the bottom of the tube until the magnetic beads are fully dispersed, placing the tube on a magnetic frame to make the magnetic beads be adsorbed to one side of the tube wall,
f. the supernatant was carefully removed, without centrifugation,
g. the SA-PMPs were washed three times with 300. mu.L of 0.5 XSSC buffer, magnetic beads were collected by magnetic adsorption on a magnetic rack each time, the supernatant was carefully removed,
h. finally resuspend SA-PMPs with 100. mu.L of 0.5 XSSC;
s1.4, capturing and washing oligo (dT) -mRNA hybrid strand, comprising:
i. adding all reaction products obtained in the step S1.1 and in the probe annealing process into the cleaned SA-PMPs, gently mixing the reaction products,
j. incubating for 10 minutes at room temperature, slightly inverting and mixing the mixture once every 1 to 2 minutes,
k. adsorbing and collecting SA-PMPs by a magnetic frame, carefully sucking the solution into a sterile 1.5mL centrifuge tube without touching magnetic beads, placing the centrifuge tube on ice for later use,
m, washing the magnetic beads by using 300 mu L of 0.1 XSSC buffer solution, flicking the bottom of the tube until the magnetic beads are fully resuspended, removing supernatant, and repeatedly washing for four times;
s1.5, eluting mRNA, comprising:
n, resuspending the SA-PMPs in step S1.4 with 100. mu.L of RNase-free water, resuspending the magnetic beads at the bottom of the flick tube,
o, adsorbing the magnetic beads by a magnetic frame, absorbing the eluted mRNA solution into a sterile centrifuge tube, simultaneously retaining the magnetic beads,
p, washing SA-PMPs with 150. mu.L of RNase-free water repeatedly once, sucking the solution into the tube in the previous step,
s1.6, purification, concentration of eluted mRNA (RNA Clean)&Concentrator TM -5)
q, buffer preparation: adding 48mL of absolute ethanol to 12mL of RNA Wash buffer,
r, adding RNA binding buffer with twice volume to each sample tube in the step S1.5 for eluting mRNA, mixing uniformly, adding absolute ethyl alcohol with the same volume, mixing uniformly, adding the mixed solution to Zymo-Spin put in a collecting tube TM In IICR Column, centrifuging at 12000g at room temperature for 1 minute, discarding the liquid in the collection tube,
s, adding 400 mu L of RNA prep buffer into an adsorption column, centrifuging for 1 minute at room temperature, discarding the liquid in a collection tube,
t, adding 700 mu LRNA wash buffer into the adsorption column,
u, adding 400 mu L of RNA wash buffer into the adsorption column, centrifuging for 1 minute at room temperature, discarding the liquid in the collection tube, centrifuging the empty tube for 1 minute at room temperature again, completely removing the wash buffer, carefully moving the column into an RNase-free centrifuge tube,
v, adding 10 mu L of RNase-free water into the adsorption column, centrifuging for 1 minute at room temperature to elute the RNA,
s1.7, taking 1 mu L of the RNA solution in the step S1.6, and detecting the nucleic acid concentration by using a Qubit 3.0;
s2, addition of A reaction without poly A tail RNA
The solution of step S1.4 contains no poly A tailRNA, rRNA and tRNA, and RNA Clean&Concentrator TM -25, carrying out the purification by using the kit,
s2.1, adding RNA binding buffer with twice volume to each sample tube, adding absolute ethyl alcohol with the same volume after mixing uniformly, adding the mixed solution to Zymo-Spin after mixing uniformly TM In IICR Column, centrifuging for 1 minute at room temperature, discarding the liquid in the collecting tube,
1) adding 400 mu L of RNA prep buffer into an adsorption column, centrifuging for 1 minute at the room temperature of 12000g, discarding the liquid in a collection tube,
2) adding 700 mu L of RNA wash buffer into an adsorption column, centrifuging for 1 minute at the room temperature of 12000g, discarding the liquid in a collection tube,
3) adding 400 mu L of RNA wash buffer into an adsorption column, centrifuging for 1 minute at the room temperature of 12000g, discarding the liquid in a collecting pipe,
4) the empty tube was centrifuged again at 12000g for 1 min at room temperature to completely remove the wash buffer. Carefully move the column to the RNase-free centrifuge tube,
5) adding 30 μ L RNase-free water into the adsorption column, centrifuging at room temperature for 1 min to elute RNA,
6) preparing an A adding reaction system as follows:
RNA solution 30. mu.L, 10 XE. coli Poly (A) Polymerase Reaction Buffer 4. mu.L (1X), ATP (10mM) 4. mu.L, E.coli Poly (A) Polymerase 2. mu.L in step 5);
7) after incubating the reaction mixture at 37 ℃ for 30 minutes, EDTA was added to a final concentration of 10mM to terminate the reaction,
8) by RNA Clean&Concentrator TM -5 kit purification of the reaction product of step 7), in particular by:
a1, adding two volumes of RNA binding buffer to each sample tube, and mixing. Adding equal volume of absolute ethyl alcohol, mixing evenly,
a2, adding the mixture to Zymo-Spin TM In IICR Column, 12000g of the solution is centrifuged for 1 minute at room temperature, the liquid in a collecting tube is discarded,
a3, adding 400 mu L of RNA prep buffer into an adsorption column, centrifuging for 1 minute at 12000g, discarding the liquid in a collection tube,
a4, adding 700 mu L RNA wash buffer into the adsorption column, centrifuging for 1 minute at 12000g, discarding the liquid in the collection tube,
a5, adding 400 mu L RNA wash buffer into the adsorption column, centrifuging for 1 minute at 12000g, discarding the liquid in the collection tube,
a6, centrifuging the empty tube again at 12000g at room temperature for 1 minute to completely remove the wash buffer,
a7, carefully transferring the adsorption column to the RNase-free centrifuge tube, adding 10. mu.L of RNase-free water to the column, centrifuging at 12,000g for 1 minute, eluting RNA,
9) taking 1 mu L of the eluted RNA solution, and detecting the concentration of nucleic acid by using a Qubit 3.0;
s3, establishing a library, comprising:
b1, RNA dosage: the total volume is 9. mu.L, the total volume is 500ng, namely the RNA products obtained in the step S1 and the step S2 are respectively subjected to library construction,
b2, preparing the following reaction system in a PCR tube:
mixing NEBNext Quick Ligation Reaction Buffer 3.0 mu L, RNA 9.0.0 mu L, RNA CS (RCS)110nM 0.5 mu L, Barcode 1/Barcode 2/Barcode 3/Barcode 4/RTAdap (RTA)1.0 mu L, T4 DNA ligase 1.5 mu L, then quickly centrifuging, quickly incubating at room temperature for 10 min, synthesizing 4 pairs of Barcode with HPLC grade according to the nucleotide sequence information of Barcode 1/Barcode 2/Barcode 3/Barcode 4, diluting the A and B components of each pair of Barcode to 10 mu M final concentration respectively with RNase-free water, mixing the A and B components of each pair of Barcode in equal volume before use;
b3, preparing a reaction system for reverse transcription, which comprises 9.0 μ L of nucleic-free water, 2.0 μ L of 10mM dNTPs, 8.0 μ L of 5 Xfirst-strand buffer, 4.0 μ L of 0.1M DTT,
b4, adding the mixed solution in the step b3 into the reaction system in the step b2, mixing evenly,
b5, adding 2 mu L SuperScript III reverse transcriptase into the reaction system, mixing evenly,
b6, setting a PCR amplification program, keeping at 50 ℃ for 50 minutes, 70 ℃ for 10 minutes and 4 ℃ respectively, carrying out PCR amplification,
b7, transferring the PCR product in the step b6 into a sterile 1.5mL centrifuge tube,
b8, resuspending Agencour RNAclean XP beads by vortex shaking, sucking 72 mu L of Agencour RNAclean XP beads into the reaction system in the step b7, uniformly mixing by a pipette, and incubating for 5 minutes at room temperature in a Hulamixer,
b9, placing the sample tube on a magnetic frame, operating on the magnetic frame, sucking the supernatant, washing the magnetic beads with 150 mu L of 70% ethanol, finally sucking 70% ethanol by a pipette,
b10, removing the sample tube from the magnetic frame, resuspending the magnetic beads in 20. mu.L of nuclease-free water, incubating at room temperature for 5 minutes,
b11, adding the 20 mu L of eluent into a sterile 1.5mL Eppendorf DNA Lobind tube, and preparing a joint connection reaction system: comprises 20.0 mu L of RNA solution in the step b10, 8.0 mu L of NEBNext Quick Ligation Reaction Buffer, 6.0 mu L of RNA Adapter (RMX) and 3.0 mu L of nuclease-free water, the components are mixed evenly and incubated for 10 minutes at room temperature,
b12, adding 40 mu L of RNAclean XP beads into a joint connection reaction system, uniformly mixing, incubating for 5 minutes in a Hula mixer at room temperature,
b13, placing the sample tube on a magnetic frame, sucking the supernatant, adding 150 mu L of wash buffer (WSB) to clean the magnetic beads, sucking the supernatant, repeating the cleaning once,
b14, adding 21 mu L of elution buffer solution (EB), resuspending magnetic beads, incubating for 10 minutes at room temperature, placing the tube on a magnetic frame, sucking supernatant into a sterile centrifuge tube, taking 1 mu L of the supernatant, and detecting the nucleic acid concentration to complete library establishment.
2. The direct RNA sequencing library construction method of claim 1, wherein: the total RNA in the step a is more than or equal to 50 mu g.
3. The direct RNA sequencing library construction method of claim 1, wherein: in the step S1.2, the cooling time of the RNA solution is more than or equal to 10 minutes.
4. A direct RNA sequencing library building method according to claim 1, wherein: the solution for capturing and washing oligo (dT) -mRNA hybrid strands in step S1.4 contains RNA without a ploy A tail.
5. The direct RNA sequencing library construction method of claim 1, wherein: the step a3, the step a4 and the step a5 are all centrifuged at room temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210250375.8A CN115044646A (en) | 2022-03-15 | 2022-03-15 | Direct RNA sequencing and database building method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210250375.8A CN115044646A (en) | 2022-03-15 | 2022-03-15 | Direct RNA sequencing and database building method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115044646A true CN115044646A (en) | 2022-09-13 |
Family
ID=83158527
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210250375.8A Pending CN115044646A (en) | 2022-03-15 | 2022-03-15 | Direct RNA sequencing and database building method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115044646A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116904445A (en) * | 2023-09-12 | 2023-10-20 | 南京诺唯赞生物科技股份有限公司 | mRNA enrichment method |
-
2022
- 2022-03-15 CN CN202210250375.8A patent/CN115044646A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116904445A (en) * | 2023-09-12 | 2023-10-20 | 南京诺唯赞生物科技股份有限公司 | mRNA enrichment method |
| CN116904445B (en) * | 2023-09-12 | 2023-12-29 | 南京诺唯赞生物科技股份有限公司 | mRNA enrichment method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113151397B (en) | Nucleic acid extraction kit for extracting virus sample based on magnetic bead method | |
| CN106591297A (en) | Magnetic bead nucleic acid extraction method | |
| CN107893100A (en) | A kind of unicellular mRNA reverse transcriptions and the method for amplification | |
| CN115044646A (en) | Direct RNA sequencing and database building method | |
| JP7248228B2 (en) | Methods and kits for construction of RNA libraries | |
| CN109762874A (en) | Nucleic acid settling agent, pregnant woman blood plasma dissociative DNA extracts kit and method | |
| CN110592200B (en) | Multiplex PCR method for improving amplification specificity and uniformity | |
| CN107354207B (en) | liquid phase hybridization capture kit based on double-stranded probe, washing kit and application thereof | |
| WO2015196752A1 (en) | A method and a kit for quickly constructing a plasma dna sequencing library | |
| CN113373201A (en) | Probe composition for preventing reverse transcription of Globin mRNA and application thereof | |
| CN112048503A (en) | Kit for extracting plant genome DNA by high-throughput rapid magnetic bead method and extraction method | |
| CN112176421A (en) | RNA library building method | |
| CN112322614A (en) | Kit for extracting blood genome DNA by paramagnetic particle method and use method thereof | |
| CN103695419B (en) | A kind of Viral nucleic acid extraction reagent | |
| CN115960885B (en) | Method and composition for extracting nucleic acid from heparin sodium sample | |
| CN114277091B (en) | Method for constructing high-quality immune repertoire library | |
| WO2020118543A1 (en) | Method for separating and/or enriching host source nucleic acid and pathogenic nucleic acid, and reagent and preparation method therefor | |
| CN116262918A (en) | Probe set, kit and application thereof, method for removing ribosomal RNA in sample and method for extracting nucleic acid in biological sample | |
| CN112481254B (en) | Method and kit for removing host DNA and enriching microorganisms by one-step method | |
| CN112195219B (en) | Noise reduction method for targeted capture of enrichment sequencing library molecules by small panel probe | |
| CN109439655B (en) | Kit and method suitable for extracting ultra-trace nucleic acid | |
| CN114350649A (en) | Nucleic acid extraction kit and nucleic acid extraction method | |
| CN117661124A (en) | Method and kit for constructing RNA library | |
| CN116162690B (en) | One-tube targeting high-throughput sequencing method | |
| CN116949156B (en) | Analysis method for detecting human T cells in general way based on nucleic acid variants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |