CN113025688A - Method for detecting full-length non-poly (A) transcript based on customized nanopore direct RNA sequencing reverse transcription linker - Google Patents
Method for detecting full-length non-poly (A) transcript based on customized nanopore direct RNA sequencing reverse transcription linker Download PDFInfo
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Abstract
The invention discloses a construction method and application of a sequence specificity direct RNA sequencing library based on Oxford Nanopore Technologies, belonging to the technical field of third-generation high-throughput sequencing. The construction method comprises the following steps: extracting total RNA and controlling quality; removal of rRNA from total RNA and quality control; designing the design, construction and quality control of the sequence-specific reverse transcription linker; direct RNA sequencing library construction based on Oxford Nanopore Technologies; and (3) detecting sequence-specific RNA. The present method redesigns ssRTA and applies it to Oxford Nanopore Technologies based direct RNA sequencing libraries, mainly for the determination of linear transcripts without a ploy (a) tail, which was not possible with any previous procedure.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a construction method and application of a sequence specificity direct RNA sequencing library based on Oxford Nanopore Technologies.
Background
Direct RNA sequencing based on Oxford Nanopore Technologies is the only technology capable of directly sequencing natural RNA molecules in full length and single molecules at present, the standard library construction process is that reverse transcription joints (RTA) containing Oligo d (T) are connected to all RNA molecules to be tested, the 3' tail ends of the RNA molecules are provided with Poly (A) structures, then reverse transcription is carried out to generate a first cDNA chain, and then sequencing joints (RMX) are connected to the tail ends of the RTA to complete library construction. However, the current method is limited to the sequence of RTA, and can only sequence RNA molecules with a structure of Poly (A) at the 3' end.
The invention designs and develops a sequence specificity reverse transcription linker (ssRTA) based on Oxford Nanopore Technologies direct RNA sequencing technology, realizes the full-length single-molecule direct sequencing of RNA without 3' Poly (A) structure, and enriches the types of transcripts capable of being detected by direct RNA sequencing.
Disclosure of Invention
The invention provides a construction method and application of a sequence specificity direct RNA sequencing library based on Oxford Nanopore Technologies.
In order to achieve the purpose, the invention adopts the following technical scheme.
A construction method and application of a sequence specificity direct RNA sequencing library based on Oxford Nanopore Technologies are characterized in that the method comprises the following steps: (1) extracting total RNA and controlling quality; (2) removal of rRNA from total RNA and quality control; (3) design, construction and quality control of sequence specific reverse transcription adaptors (ssRTA); (4) direct RNA sequencing library construction based on Oxford Nanopore Technologies; (5) and (3) detecting sequence-specific RNA. Optionally, the total RNA sample is derived from a plant, animal or human.
In the step (1), the extraction and quality control of total RNA comprises:
the moso bamboo seedlings were cleaned as shown in fig. 1.
Further, total RNA of the sample is extracted by a TIANGEN RNAprep Pure polysaccharide polyphenol plant total RNA kit.
Further, the degree of degradation of the total RNA samples was examined by 1.0% agarose gel electrophoresis (6V/cm), as shown in FIG. 2.
Further, the total RNA samples were accurately quantified using a Qubit fluorometer.
Further, the purity of the total RNA samples was measured using a Nanodrop 2000c spectrophotometer and characterized as A260/280 and A260/230.
Further, the RIN values of total RNA samples were determined using an Agilent 2100 bioanalyzer and subsequent experiments were performed using samples with RIN values > 8.0, as shown in fig. 3.
The step (2) of removing rRNA from total RNA and controlling quality comprises:
by RiboMinusTMPlant kit for RNA-seq (ThermoFisher, Cat. number A1083808) removed rRNA from the total RNA sample to give a Ribo-RNA sample.
Further, the rRNA removal effect was examined by 1.0% agarose gel electrophoresis (6V/cm), as shown in FIG. 4.
And (3) designing, constructing and controlling the quality of the sequence specific reverse transcription adaptor (ssRTA):
synthesis of 2 primers, SEQ ID No.1:
5’ - /PHOS/ GGCTTCTTCTTGCTCTTAGGTAGTAGGTTC - 3’，
SEQ ID NO.2:
5’ - GAGGCGAGCGGTCAATTTTCCTAAGAGCAAGAAGAAGCCNNNNNNNNNN-3', the underlined part is the 20 bp complementary region of 2 primers;
further, 2 pieces of primer dry powder are dissolved by sterile deionized water, mixed uniformly in equal quantity, placed in a PCR instrument for 10 min at 95 ℃, and then slowly annealed to 30 ℃ at the speed of 0.1 ℃/s.
Further, 5. mu.L of the prepared ssRTA was incubated at 98 ℃ for 2 min with a PCR instrument and immediately placed on ice, and the change in absorbance at 260 nm of the ssRTA before and after incubation was measured with a Nanodrop 2000c spectrophotometer, as shown in FIG. 5.
The above step (4), direct RNA sequencing library construction based on Oxford Nanopore Technologies includes:
sequence specific reverse transcription linkers (ssRTA) were ligated to the ends of Ribo-RNA using a rapid ligation buffer (NEB, B6058) and T4 DNA ligase (NEB, M0202).
Further, the first strand cDNA was synthesized by reverse transcription using SuperScript III reverse transcriptase (ThermoFisher, 18080044).
Further, a direct RNA sequencing kit (ONT, SQK-RNA 002) and T4 DNA ligase were used to ligate a sequencing linker (RMX) to the end of the RNA sample to be tested.
Furthermore, the library was carried on R9.4.1 sequencing chip (ONT), sequenced on the MinION MK 1B device (ONT), and the direct RNA sequencing process was controlled using MinKNOW software (ONT, version: 19.06.7), as shown in FIG. 6.
The invention has the advantages that:
based on Oxford Nanopore Technologies direct RNA sequencing technology, a sequence-specific reverse transcription linker (ssRTA) is designed and developed, full-length single-molecule direct sequencing of RNA without a 3' Poly (A) structure is realized, and the information of the type of a direct RNA sequencing transcript is enriched.
Description of the drawings:
FIG. 1 shows a Phyllostachys pubescens seedling.
FIG. 2 shows the results of detecting the degree of degradation of total RNA samples by 1.0% agarose gel electrophoresis; wherein M represents Marker; lanes 1, 2, and 3 are three different technical replicates of the same material powder, respectively; 28 s and 18 s rRNA bands were clearly visible, and no smaller bands or streaking were observed with the naked eye, indicating that the total RNA samples were not degraded.
FIG. 3 shows the results of the RIN value determination of total RNA samples; wherein, the left side is an RNA fragment size peak diagram obtained by an Agilent 2100 bioanalyzer, and the right side is an RNA agarose gel electrophoresis simulation diagram obtained by the Agilent 2100 bioanalyzer.
FIG. 4 shows rRNA removal by 1.0% agarose gel electrophoresis; where M represents Marker, Ribo + represents total RNA sample, Ribo-represents RNA sample after rRNA removal, 28 s and 18 s rRNA bands of rRNA-removed RNA sample were significantly reduced.
FIG. 5 shows the hyperchromic effect of the high temperature denatured ssRTA due to strand unraveling, where A260 is significantly enhanced over the non-high temperature denatured ssRTA, and thus the double-stranded ssRTA can be used for downstream library construction.
FIG. 6 is a schematic diagram showing the running status of each sequencing channel in real time by MinKNOW software; 458 channels are in a sequencing state, 44 channels are in a state to be sequenced, 2 channels are in a blocking state, 3 channels are in an inactivation closing state, and 5 channels are in an unclassified state which is not the four states.
FIG. 7 shows the effect of detecting circular RNA in bamboo seedlings based on the method of the present invention.
Detailed Description
The following raw materials are Mao bamboo (Phyllostachys edulis) The detailed description of the embodiments of the present invention is given by way of example of seedling, which is only for the purpose of illustrating the present invention and is not to be construed as limiting the scope of the present invention. In the examples, the experimental equipment and reagents are commercially available.
Example 1 extraction and quality control of Total RNA
(1) Extraction of total RNA of moso bamboo seedlings
Bamboo seedlings were treated to a clean state as shown in fig. 1 for total RNA extraction. Total RNA was extracted using a TIANGEN RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (Cat. number DP 441). All EP tubes, pipette tips were RNase-free.
a. Wrapping the material with tinfoil, quick freezing with liquid nitrogen, quickly filling into liquid nitrogen precooled tissue grinding jar (QIAGEN, Cat. number 69985) after 10 min, and fully grinding for 90 s with high throughput tissue grinder (QIAGEN, Cat. number 85300) under 30 times/s vibration frequency.
b. About 100 mg of the powder was transferred to a 1.5 mL RNase-free EP tube, 500. mu.L of SL (containing 10% beta-mercaptoethanol) was added thereto, and immediately mixed by vigorous shaking for 20 seconds by a vortex shaker.
c. Centrifuge at 13000 rpm for 2 min.
d. The supernatant was transferred to a CS filtration column (filter column placed in collection tube), centrifuged at 13000 rpm for 2 min, and the supernatant in the collection tube was pipetted into a new RNase-free EP tube (about 350. mu.L, exact volume measured with pipette) to avoid contact of the pipette tip with the pellet.
e. Slowly adding 0.4 times of the volume of the supernatant into absolute ethyl alcohol, gently mixing uniformly (white flocculent precipitate can be observed to slowly appear in the mixing process), transferring all the contents in the tube into a CR3 adsorption column, centrifuging at 13000 rpm for 30 s, discarding the waste liquid in the collection tube, and returning the CR3 adsorption column to the collection tube.
f. 350 μ L of RW1 deproteinized solution was added to the adsorption column, centrifuged at 13000 rpm for 30 s, the waste liquid in the collection tube was discarded, and the CR3 adsorption column was returned to the collection tube.
g. Mixing 70 mu L of RDD solution and 10 mu L of DNase I storage solution to prepare DNase I working solution, adding the DNase I working solution into the center of a CR3 adsorption column, and incubating for 15-20 min at room temperature.
h. And f, repeating the step f.
i. Add 500. mu.L RW rinse to the adsorption column, centrifuge at 13000 rpm for 30 s, discard the waste from the collection tube, and return the CR3 adsorption column to the collection tube.
j. And (e) repeating the step i.
k. Performing air separation at 13000 rpm for 2 min, placing CR3 adsorption column into a new RNase-free EP tube, dripping 35-55 μ L RNase-free water into the center of the adsorption membrane, standing at room temperature for 2 min, and centrifuging at 13000 rpm for 1 min.
l, adding the RNA solution obtained in the step k into the center of a CR3 adsorption column, carrying out redissolution for 1 time, standing at room temperature for 2 min, centrifuging at 13000 rpm for 5 min to obtain a final total RNA solution, and quickly placing on ice for later use.
(2) Agarose gel electrophoresis detection of total RNA sample degradation degree
The total RNA solution (1. mu.L) was aspirated, diluted 5-fold with RNase-free water, and mixed with 6 × loading buffer (1. mu.L), followed by agarose gel electrophoresis at 6V/cm for 25 min, with the quality control results shown in FIG. 2.
(3) Precise quantification of total RNA samples by a Qubit fluorometer
Total RNA samples were quantitated accurately using the Qubit-round RNA HS Assay Kit (ThermoFisher, Cat. number Q32855). The upper limit of the quantitative concentration was 100 ng/. mu.L, and total RNA was diluted with RNase-free water before starting the following procedure.
a. Set the required number of 0.5 mL assay tubes for the standards and samples. The Qubit ™ RNA HS Assay Kit requires 2 standards.
Specifically, only thin-walled, clear, 0.5 mL tubes were used. Useful Assay tubes include the Qubit-antibody Tube (ThermoFisher, Cat. number Q32856), AxygenTMPCR-05-C tube (Axygen, Cat. number 10011-830). Preferably, a Qubit-antibody Tube is used.
b. The tube caps are marked.
In particular, the sides of the tube are not marked, which may interfere with the sample reading. Calibration of the Qubit fluorometer requires that the standards be inserted into the instrument in the correct order.
c. By applying at the QubitTMDiluting the Qubit in RNA HS bufferTMRNA HS reagent 1: 200 to prepare the QubitTMA working solution. Prepare the Qubit each timeTMIn the case of the working solution, a clean plastic tube is used without mixing the working solution into a glass container.
In particular, the final volume in each tube must be 200 μ L. Each standard tube requires 190 μ L of QubitTMWorking solution, each sample tube required 180-. Prepare enough qubitsTMWorking solution to accommodate all standards and samples.
d. Add 190. mu.L of Qubit to each tube for standardsTMA working solution.
e. mu.L of each of the qubits was addedTMStandards were added to the appropriate tubes and vortex mixed for 2-3 seconds. Care was taken not to generate bubbles.
In particular, careful pipetting is used to ensure that at 190. mu.L of the QubitTM Working solution addition 10 μ L of each kind of QubitTMStandards are of critical importance.
f. Will QubitTMThe working solution was added to each tube so that the final volume in each tube after addition of the sample was 200 μ L.
In particular, the sample may be 1-20. mu.L. Add a corresponding volume of Qubit in each cuvetteTMWorking solution: 180-.
g. Each sample was added to a volume containing the correct volume of the QubitTMWorking solution was placed in the assay tube and then vortex mixed for 2-3 seconds. The final volume in each tube should be 200 μ L.
h. All assay tubes were incubated for 2 minutes at room temperature in the dark.
i. Click on select RNA samples on the Qubit fluorometer display, further select RNA: high sensitivity is used as the measurement type, and the screen displays "Standards".
In particular, if a standard curve construction has been performed for the selected analysis mode, the instrument will prompt a choice between constructing a new standard curve and performing a sample measurement with the previous standard curve. If the previous standard curve is to be used, press No and jump to step m. Otherwise, continue to step j.
j. On the standard screen, the standard is measured as Yes.
k. The tube containing Standard # 1 was inserted into the sample chamber, the lid closed, and then Read. After completion of the reading (about 3 seconds), the Standard # 1 was taken out.
Insert the tube containing the standard # 2 into the sample chamber, close the lid, and press Read. After the reading was complete, the # 2 standard was removed. The calibration is complete and the screen displays "Sample".
m. insert sample tube into sample chamber, close lid, then press Read. After the reading was complete (about 3 seconds), the sample tube was removed. The instrument displays the quantitative results under the "Sample" option.
n. repeating step m until quantification is completed for all samples.
(4) Nanodrop 2000c spectrophotometer detection of total RNA sample purity
And (3) opening the Nanodrop 2000c spectrophotometer determination software, clicking 'Nucleic Acid', selecting an RNA determination mode after the self-test of the instrument, and determining the purity of the total RNA sample by using RNase-free water as a blank control.
Preferably, the total RNA sample has an A260/280 of about 2.0 and an A260/230 of 2.0-2.2.
Generally, a 260/280 below 2.0 indicates the potential for DNA contamination and below 1.8 indicates the potential for protein or phenolic contamination. A260/230 below 2.0 indicates that additional purification is required for the total RNA sample.
(5) Total RNA sample RIN value determined by Agilent 2100 bioanalyzer
mu.L of total RNA samples were analyzed using an Agilent 2100 bioanalyzer and an RNA 6000 Nano Kit (Agilent, Cat. number 5067- & 1511). The results are shown in FIG. 3.
Preferably, samples with RIN values > 8.0 are taken for subsequent experiments.
Example 2 removal of rRNA from Total RNA and quality control
(1) Removal of rRNA from Total RNA samples
By RiboMinusTMPlant kit for RNA-seq (ThermoFisher, Cat. number A1083808) total rRNA removal from total RNA samples.
a. Hybridization of probes
1) Setting 2 water baths, 1 at 70 deg.C and 1 at 37 deg.C.
2) In a sterile 1.5 mL EP tube with RNase-free the following components were added:
3) incubate at 70 ℃ for 5 min in a water bath to denature the RNA.
4) Slowly cooling the denatured total RNA sample in a water bath kettle at 37 ℃ for 30 min, and flicking once every 10 min.
5) Preparing magnetic beads during the temperature reduction process of the total RNA sample.
b. Preparing magnetic beads
1) Resuspending the RiboMinus thoroughly by vortexingTMMagnetic beads.
2) Pipette 750. mu.L of magnetic beads into a sterile and RNase-free 1.5 mL EP tube (tube No. 1).
3) The tube was placed on a magnetic stand for 1 min, and after the beads had accumulated, the supernatant was aspirated.
4) The beads were washed by adding 750 μ L sterile DEPC water to the tube and resuspended by slow vortexing.
5) Put the tube back to the magnetic stand, wait for 1 min before the beads aggregate, and discard the supernatant.
6) Repeat step 4), 5).
7) Add 750. mu.L Hybirdization Buffer to the tube, resuspend the beads by vortexing slowly, then transfer 250. mu.L to a new sterile 1.5 mL EP tube (tube 2) with RNase-free, and place tube 2 in a 37 ℃ water bath until use.
8) Tube 1 was placed on a magnetic stand for 1 min, and after 500. mu.L of magnetic beads had accumulated, the supernatant was aspirated.
9) Pipette 200. mu.L of Hybirdization Buffer to resuspend No.1 tube beads, and place No.1 tube in 37 ℃ water bath all the time until use.
c. Removal of rRNA
1) Taking out the total RNA sample incubated in the water bath kettle at 37 ℃ by slow cooling, and placing the total RNA sample on a micro centrifuge for brief centrifugation for 2 s.
2) Transfer 120 μ L of total RNA sample hybridized to probe to tube No.1 and mix the sample and magnetic beads by pipetting.
3) The tube No.1 is incubated in a water bath at 37 deg.C for 15 min, during which time the tube is flicked and mixed once every 5 min.
4) Taking out the No. 2 tube from the water bath kettle at 37 ℃, placing the tube on a magnetic frame for 1 min until the magnetic beads are aggregated, and sucking and removing the supernatant.
5) The incubated tube No.1 was removed from the 37 ℃ water bath, placed on a magnetic rack for 1 min for aggregation of the magnetic beads, the supernatant was carefully pipetted about 320. mu.L into the tube No. 2, pipetted gently to resuspend the magnetic beads and mix the components.
6) The tube No. 2 is incubated in a water bath at 37 deg.C for 15 min, during which time the tube is flicked and mixed once every 5 min.
7) The incubated tube 2 was removed from the 37 ℃ water bath, placed on a magnetic rack for 1 min for magnetic beads to aggregate, and the supernatant carefully pipetted into a fresh sterile 1.5 mL RNase-free EP tube (tube 3).
d. Collecting RNA after rRNA removal
1) Adding the following components into a No. 3 tube, adding one component for each component, sucking, beating and uniformly mixing, and then adding the next component:
2) mixing the components in tube No. 3, and standing in refrigerator at-80 deg.C for at least 30 min. Preferably, it is left to stand overnight.
3) The tube 3 after the completion of the standing was taken out, 12000g was centrifuged at 4 ℃ for 20 min, and the supernatant was carefully discarded.
4) Add 500. mu.L of 70% ethanol pre-cooled to 20 ℃ into tube 3, gently pipette and resuspend the RNA pellet.
5) Tube 3 was centrifuged at 12000g at 4 ℃ for 20 min and the supernatant carefully discarded.
6) Repeat step 4), 5).
7) Air-dry tube No. 3 at room temperature, dissolve RNA sample (Ribo-RNA) completely after about 5 min with 10-30 μ L DEPC water to remove rRNA.
8) The Ribo-RNA sample was quantified precisely by the method described in example 1- (3), and then stored in a refrigerator at-80 ℃ for further use.
(2) Agarose gel electrophoresis detection of rRNA removal effect
After equal mass of total RNA sample (Ribo + RNA) and Ribo-RNA sample before rRNA removal are respectively sucked and mixed with 6 × loading buffer, agarose gel electrophoresis is carried out for 25 min under the voltage of 6V/cm, the quality control result is shown in figure 4, the 28 s rRNA band and the 18 s rRNA band of the Ribo + RNA sample are cleaned and are not observed, and the 28 s rRNA band and the 18 s rRNA band of the Ribo-RNA are observed.
Example 4 design, construction and quality control of sequence specific reverse transcriptase linkers (ssRTAs)
(1) Design of ssRTA
Designing 2 primers:
SEQ ID NO.1:
5’ - /PHOS/ GGCTTCTTCTTGCTCTTAGGTAGTAGGTTC - 3’
SEQ ID NO.2:
5’ - GAGGCGAGCGGTCAATTTTCCTAAGAGCAAGAAGAAGCCNNNNNNNNNN - 3’
the regions complementary to the 2 primers are underlined.
(2) Construction of ssRTA
Dissolving 2 pieces of primer dry powder in sterile deionized water, mixing uniformly in equal amount, placing in a PCR instrument, keeping at 95 ℃ for 10 min, and then slowly annealing to 30 ℃ at the speed of 0.1 ℃/s.
(3) Quality control of ssrTA
mu.L of the prepared ssRTA was taken, incubated for 2 min at 98 ℃ with a PCR instrument, immediately placed on ice, and then the change in absorbance at 260 nm of the ssRTA before and after incubation was measured with a Nanodrop 2000c spectrophotometer. As shown in FIG. 5, the nucleic acid molecule hyperchromic effect of the high temperature denatured ssRTA due to double strand unwinding, A260, is significantly improved compared to the non-high temperature denatured ssRTA, so that the double-stranded ssRTA can be used for downstream library construction.
Example 4 Oxford Nanopore Technologies based direct RNA sequencing library construction
(1) Ligation of sequence-specific reverse transcription linkers (ssRTA) to Ribo-RNA termini
a. 500 ng Ribo-RNA was transferred to a sterile nuclease-free 1.5 mL EP tube, adjusted to a final volume of 9.0. mu.L with nuclease-free water (ThermoFisher, Cat. number AM 9937), and mixed by inversion.
b. The reagents were added sequentially to a 0.2 mL thin-walled PCR tube, pipetted and mixed well.
c. Incubate at room temperature for 10 min.
(2) Reverse transcription to synthesize the first chain
a. The following components were mixed well in a sterile nuclease-free 1.5 mL EP tube.
b. Add 23.0. mu.L of the above solution to the 0.2 mL thin-walled PCR tube from step (1) -b, pipette and mix well.
c. Add 2.0. mu.L SuperScript III reverse transcriptase (ThermoFisher, 18080044) and mix by pipetting.
d. The reverse transcription reaction system of 40.0. mu.L is placed in a PCR instrument under the conditions of 50 ℃ for 50 min and 70 ℃ for 10 min, and is taken out and immediately placed on ice after completion.
e. After completion of the reverse transcription reaction, it was transferred to a sterile nuclease-free EP tube 1.5 mL, mixed well with 72.0. mu.L of RNAclean XP magnetic beads (Beckman Coulter, Cat. number A63987), and incubated on a mixer at room temperature for 5 min.
f. Placing a 1.5 mL EP tube on a magnetic frame, after the magnetic bead aggregation liquid is clarified, absorbing and discarding the supernatant, washing the magnetic beads for 2 times by using freshly prepared 70% ethanol, centrifuging the 1.5 mL EP tube for 5 s, placing the EP tube back on the magnetic frame, after the magnetic beads are aggregated, and absorbing and discarding any residual liquid.
g. Resuspend the beads with 20.0 μ L nuclease-free water, incubate at room temperature for 5 min, place back on the magnetic rack, wait for the beads to aggregate, transfer the supernatant to a new sterile nuclease-free 1.5 mL EP tube.
(3) Sequencing linker (RMX) ligation
a. The following components were mixed well in a sterile nuclease-free 1.5 mL EP tube, pipetted and mixed well.
b. Incubate at room temperature for 10 min.
c. Mix well with 40.0 μ L RNAClean XP beads and incubate for 5 min at room temperature on the mixer.
d. Placing the 1.5 mL EP tube on a magnetic frame, clarifying the liquid of magnetic bead aggregation, removing the supernatant, washing the magnetic beads with 150.0 μ L WSB (ONT, SQK-RNA 002) for 2 times, centrifuging the 1.5 mL EP tube for 5 s, placing the tube back on the magnetic frame for magnetic bead aggregation, and removing any residual liquid.
e. Resuspend the magnetic beads with 21.0. mu.L ELB (ONT, SQK-RNA 002), incubate at room temperature for 10 min, place back on the magnetic rack, after the magnetic beads aggregate, transfer the supernatant to a new sterile nuclease-free 1.5 mL EP tube, and place on ice for use.
f. The RNA library was accurately quantified by the method described in example 1- (3), preferably about 200 ng.
(4) Direct RNA sequencing by machine
The R9.4.1 sequencing chip was mounted to the MinION MK 1B instrument with USB 3.0 and above connected interface, the RNA library was loaded to the R9.4.1 sequencing chip, sequencing parameters were set and sequencing progress was controlled using MinKNOW software (version: 19.06.7).
a. Click "New experiment" in MinKNOW UI to create a New sequencing experiment.
b. The Experiment and sample names are set in the Experiment tab.
c. And setting the model of the library building Kit in the Kit tab.
d. And setting to start real-time base recognition in a basal tab.
e. Sequencing experiment Run times were set in the Run Option tab.
f. And setting a data storage path in the Output tab.
g. Click "Start run" to Start the sequencing experiment.
The operating status of each sequencing channel can be monitored during sequencing, as shown in FIG. 6.
FIG. 7A shows the number of circular RNAs containing m6A modifications. The outer circle represents the number of all circular RNAs in the sample detected based on the ssRTA method (428), the middle circle represents the number of circular RNAs that may have RRACH motif (425), and the inner circle represents the number of circular RNAs containing m6A modifications derived from the original signal of sequencing (46). The upper left part of FIG. 7B shows that m6A motif RRACH is enriched around the reverse splicing site, the lower left part of FIG. 7B shows the distance between RRACH motif and either the 5 'reverse splicing site or the 3' reverse splicing site, and the right part of FIG. 7B shows an example, an m6A site of the PH02Gene43295 Gene, which is close to the reverse splicing site.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> detection of full-length non-poly (A) transcripts based on custom-made nanopore direct RNA sequencing reverse transcription linker
Method
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence
<400> 1
ggcttcttct tgctcttagg tagtaggttc 30
<210> 2
<211> 49
<212> DNA
<213> Artificial sequence
<400> 2
gaggcgagcg gtcaattttc ctaagagcaa gaagaagccn nnnnnnnnn 49
Claims (6)
1. A method for constructing a sequence-specific direct RNA sequencing library based on Oxford Nanopore Technologies, said method comprising the steps of: (1) extracting total RNA and controlling quality; (2) removal of rRNA from total RNA and quality control; (3) designing, constructing and controlling the quality of the sequence-specific reverse transcription adapter ssRTA; (4) direct RNA sequencing library construction based on Oxford Nanopore Technologies; (5) and (3) detecting sequence-specific RNA.
2. The method of claim 1, wherein: the extraction and quality control of the total RNA in the step (1) comprise the following steps:
cleaning the moso bamboo seedlings;
extracting total RNA of a sample by using a TIANGEN RNAprep Pure polysaccharide polyphenol plant total RNA kit;
detecting the degradation degree of the total RNA sample by using 1.0% agarose gel electrophoresis;
accurately quantifying the total RNA sample with a Qubit fluorometer;
detecting the purity of the total RNA sample by using a Nanodrop 2000c spectrophotometer and characterizing by A260/280 and A260/230;
the RIN values of total RNA samples were determined with an Agilent 2100 bioanalyzer and subsequent experiments were performed with samples having RIN values > 8.0.
3. The method of claim 1, wherein: the step (2) of removing rRNA from total RNA and controlling quality comprises the following steps: by RiboMinusTMThe Plant kit for RNA-seq removes rRNA from the total RNA sample to obtain a Ribo-RNA sample; the rRNA removal effect was examined by 1.0% agarose gel electrophoresis (6V/cm).
4. The method of claim 1, wherein: the design, construction and quality control of the sequence specific reverse transcription linker ssRTA in the step (3) comprise the following steps:
design of ssRTA: synthesis of 2 primers:
SEQ ID NO.1: 5’ - /PHOS/ GGCTTCTTCTTGCTCTTAGGTAGTAGGTTC - 3’；
SEQ ID NO.2:
5’ - GAGGCGAGCGGTCAATTTTCCTAAGAGCAAGAAGAAGCCNNNNNNNNNN-3', the underlined part is the 20 bp complementary region of 2 primers;
construction of ssRTA: dissolving 2 pieces of primer dry powder by using sterile deionized water, mixing uniformly in equal volume, placing in a PCR instrument, keeping the temperature at 95 ℃ for 10 min, and then slowly annealing to 30 ℃ at the speed of 0.1 ℃/s;
quality control of ssRTA: mu.L of the prepared ssRTA was taken, incubated for 2 min at 98 ℃ with a PCR instrument, immediately placed on ice, and then the change in absorbance at 260 nm of the ssRTA before and after incubation was measured with a Nanodrop 2000c spectrophotometer.
5. The method of claim 1, wherein: the Oxford Nanopore Technologies-based direct RNA sequencing library construction of step (4) comprises the following steps:
ligating sequence-specific reverse transcription linker ssRTA to the ends of Ribo-RNA using quick ligation buffer and T4 DNA ligase;
synthesizing a first cDNA chain by reverse transcription of SuperScript III reverse transcriptase;
connecting a sequencing joint to the tail end of the RNA sample to be detected by using a direct RNA sequencing kit and T4 DNA ligase;
the library was loaded with R9.4.1 sequencing chips, sequenced on a MinION MK 1B instrument, and the progress of direct RNA sequencing was controlled using MinKNOW software.
6. Use of the library constructed by the method of claim 1 for full-length single-molecule direct sequencing of RNA that does not have the 3' poly (a) structure.
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| CN113718023A (en) * | 2021-08-23 | 2021-11-30 | 普瑞基准生物医药(苏州)有限公司 | Prokaryotic ribonucleic acid direct sequencing method based on bovine nanopore sequencing technology |
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