CN114645074A - Single bud micro RNA library building method - Google Patents
Single bud micro RNA library building method Download PDFInfo
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Abstract
The invention discloses a single flower bud micro RNA library construction method, which is based on Tn5 transposase to carry out RNA/DNA heterozygous chain fragmentation and simultaneously make two ends of cDNA provided with joints, thereby omitting the tedious operation steps of RNA interruption, double-chain synthesis and the like required in the conventional transcriptome library construction technology, directly putting total RNA into a library construction without mRNA enrichment in the whole process, greatly reducing the initial template input amount, and reducing a plurality of tedious steps such as the sample loss caused by RNA fragmentation, single-chain synthesis, double-chain synthesis, end repair, joint connection and the like through one-step enzymatic reaction, greatly reducing the total RNA requirement amount in the whole process, and saving at least 2 hours.
Description
Technical Field
The invention relates to the technical field of sequencing library construction, in particular to a method for constructing a transcriptome library by using trace RNA of a single plant bud.
Background
With the development of high-throughput sequencing technologies, many scientific studies have made breakthrough progress. Transcriptome sequencing is one of the most widely used high throughput sequencing technologies at present, and is capable of studying gene function from the whole genome at the level of transcription, revealing key molecular mechanisms in the development of specific biological processes.
At present, the preparation of a plant routine transcriptome sequencing library usually needs at least 100ng of RNA starting amount, however, in practical research experiments, the acquisition of plant samples is limited by a plurality of conditions, and if the research aims at a single nutrition or reproductive organ at a specific development stage, the material drawing of the experiments is difficult to obtain enough RNA for routine transcriptome library construction sequencing, thereby limiting the key research of a plurality of plants related to the growth and development stage process.
Therefore, the invention provides a method for constructing a transcriptome library by trace RNA, and can promote the key research of the process of the plant growth and development stage.
Disclosure of Invention
The invention aims to overcome the defect that the preparation of a plant transcriptome sequencing library in the prior art needs 100ng of RNA initial amount, and provides a single-flower-bud micro RNA library construction method, which can obtain a transcriptome library with higher quality by using the RNA initial amount of a single plant flower bud as low as 100 ng.
In order to achieve the purpose, the invention adopts the following technical scheme:
a single bud micro RNA library building method comprises the following steps:
pTXB1-Tn5 purification step: transformation of the pTXB1-Tn5 plasmid into competent E.coli Transetta DE 3; inoculating a single colony into an LB culture medium added with antibiotics, and culturing until OD600 is 0.9; adding 0.25mM isopropyl-beta-D-thiogalactoside for induction expression until OD600 is 3.0, and centrifuging the bacteria liquid after induction expression to prepare thalli; adding HEGX lysate and protease inhibitor, and cracking to release protein; centrifuging the cracked product, taking the supernatant, adding a PEI solution, centrifuging to remove precipitates, and performing chromatography on the obtained supernatant; then cracking the chromatographic column to realize the cutting of the pTXB1-Tn5 plasmid from the intron; collecting liquid containing target protein to obtain a Tn5 protein unconcentrated solution; then dialyzing by using an ultrafiltration concentration column to obtain Tn5 transposase;
RNA extraction step: extracting RNA from a single flower bud of a plant;
RNA reverse transcription step: adding oligo-dTGN primer and dNTP mixture into the solution dissolved with RNA to prepare RNA sample solution for later use; preparing a reverse transcription reaction system, wherein the reverse transcription reaction system comprises SuperScript II reverse transcriptase, an RNase inhibitor, a SuperScript II first chain buffer solution, dithiothreitol, betaine, magnesium chloride and template switching oligonucleotide; adding the RNA sample solution into a reverse transcription reaction system, and running a PCR reaction to obtain an RNA/DNA heterozygous product;
tn5 transposition complex assembly and purification steps: taking functional chimeric terminal oligonucleotide and adaptor A, taking functional chimeric terminal oligonucleotide and adaptor B to anneal step by step respectively to form double chains, then adding Tn5 transposase respectively to incubate to obtain transposome A and transposome B, mixing transposome A and transposome B, adding RNA/DNA hybrid product, and fragmenting in buffer;
preparing a single bud library and sequencing: the fragmented RNA was subjected to PCR amplification and then purified to obtain an out-of-pool product, which was quantified using a Qubit 2.0.
In one embodiment, the sequence of the oligo-dTGN primer is
5’-AAGCAGTGGTATCAACGCAGAGTACT30VN-3’。
In one embodiment, the template switch oligonucleotide in the reverse transcription reaction system has the sequence 5 '-AAGCAGTGGGTATCAACGCAGTACATRGG + G-3'.
In one embodiment, in the step of reverse transcription of RNA, the reverse transcription PCR reaction is performed by: at 42 ℃ for 90 min; at 50 ℃ for 2 min; 42 ℃ for 2min and 70 ℃ for 15 min.
In one embodiment, the functionally chimeric end oligonucleotide has a sequence of 5-CTGCTCTTATACACATCT-3, the linker A has a sequence of 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', and the linker B has a sequence of 5 ' -GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3.
In one embodiment, the buffer comprises 10mM Tris-hydroxymethyl aminomethane, 5mM MgCl in the Tn5 transposable complex assembling and purifying step 210% N, N-dimethylformamide, 9% PEG8000, 0.85mM ATP.
In one embodiment, in the RNA extraction step, total RNA is extracted from a single flower bud using Trizol extract, centrifuged with chloroform, centrifuged with isopropanol to precipitate RNA on the wall and bottom of the tube, washed with ethanol, air-dried to a transparent state, and dissolved in double distilled water sterilized at high temperature for further use.
In one embodiment, the step of subjecting the fragmented RNA to PCR amplification is: 100units of Superscript II and 1x Q5High-Fidelity Master Mix were added to the fragmented RNA products at 42 ℃ for 15 min; 70 degrees and 15 min; then adding 5 mu L N5XX and 5 mu L N7XX to perform PCR amplification, wherein the reaction procedure is 72 ℃ and 15 min; 30s at 98 ℃; 10 cycles of 98 deg.C for 20s, 60 deg.C for 20s, and 72 deg.C for 2 min; 72 ℃ for 5 min.
In one embodiment, after PCR amplification of fragmented RNA, VAHTS DNA Clean Beads were used to amplify the RNA at a rate of 1:1, purifying.
In one embodiment, the single flower buds taken are litchi or single flower buds of Ardisia japonica.
Compared with the prior art, the invention has the beneficial effects that:
the single-bud micro RNA library building method established by the invention can complete library building only by a single bud of a plant. The single-bud micro RNA library construction method is based on Tn5 transposase to segment RNA/DNA heterozygosis chains and simultaneously bring connectors at two ends of cDNA, so that the tedious operation steps of RNA breaking, double-strand synthesis and the like required in the conventional transcriptome library construction technology can be omitted, mRNA enrichment is not required in the whole process, total RNA is directly put into a library construction, the initial template input amount is greatly reduced, multiple tedious steps such as the loss of samples caused by RNA fragmentation, single-strand synthesis, double-strand synthesis, end repair, connector connection and the like are also reduced through one-step enzymatic reaction, the total RNA requirement in the whole process is greatly reduced, and at least 2 hours are saved.
The single-bud micro RNA database building method realizes the transcriptome database building of a micro plant sample, greatly simplifies the database building process and greatly reduces the total amount of the required sample at the same time compared with the current commonly used transcriptome sequencing database building method, and can realize the dynamic expression change of certain specific tissues or organs which are not identified by naked eyes in the forming process.
Drawings
The invention is further illustrated by means of the attached drawings, but the embodiments in the drawings do not constitute any limitation to the invention, and for a person skilled in the art, other drawings can be derived on the basis of the following drawings without inventive effort.
FIG. 1 is an SDS-PAGE analysis of the purification process of Tn5 protein according to the present invention;
FIG. 2 is a flow chart of the single bud microRNA library construction method of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict. Except as specifically noted, the materials and equipment used in this example are commercially available. Examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary only for the purpose of explaining the present application and are not to be construed as limiting the present application.
In the description of the present application, it is to be understood that the terms "upper", "lower", "front", "back", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present application and simplifying the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present application. In the description of the present application, "a plurality" means two or more unless specifically stated otherwise.
In the description of the present application, it should be noted that unless otherwise specifically stated or limited, the terms "connected," "communicating," and "connected" are to be construed broadly, e.g., as meaning a fixed connection, a connection through an intervening medium, a connection between two elements, or an interaction between two elements. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art as the case may be.
The terms "first," "second," and the like in the description and in the claims of the present application and in the above-described drawings are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The single bud micro RNA library construction method provided by the embodiment is characterized in that RNA is extracted from a single bud of a plant, then RNA/DNA hybrid chains are fragmented by adopting Tn5 transposase, and two ends of cDNA are provided with joints, so that the complicated operation steps of RNA interruption, two-chain synthesis and the like in the conventional transcriptome library construction technology are omitted, mRNA enrichment is not needed in the whole process, total RNA is directly put into a library construction, the total RNA requirement of the whole process is greatly reduced, and at least 2 hours are saved. The method comprises the following specific steps:
pTXB1-Tn5 purification step: transformation of the pTXB1-Tn5 plasmid into competent E.coli Transetta DE 3; inoculating a single colony into an LB culture medium added with antibiotics, and culturing until OD600 is 0.9; adding 0.25mM isopropyl-beta-D-thiogalactoside for induction expression until OD600 is 3.0, and centrifuging the bacteria liquid after induction expression to prepare thalli; adding HEGX lysate and protease inhibitor, and cracking to release protein; centrifuging the cracked product, taking the supernatant, adding a PEI solution, centrifuging to remove precipitates, and performing chromatography on the obtained supernatant; then cracking the chromatographic column to realize the cutting of the pTXB1-Tn5 plasmid from the intron; collecting liquid containing target protein to obtain a Tn5 protein unconcentrated solution; then dialyzing by using an ultrafiltration concentration column to obtain Tn5 transposase;
RNA extraction step: extracting RNA from a single flower bud of a plant;
RNA reverse transcription step: adding oligo-dTGN primer and dNTP mixture into the solution dissolved with RNA to prepare RNA sample solution for later use; preparing a reverse transcription reaction system, wherein the reverse transcription reaction system comprises SuperScript II reverse transcriptase, an RNase inhibitor, a SuperScript II first chain buffer solution, dithiothreitol, betaine, magnesium chloride and template conversion oligonucleotide; adding the RNA sample solution into a reverse transcription reaction system, and operating PCR reaction to obtain an RNA/DNA hybrid product;
tn5 transposition complex assembly and purification steps: taking the functional chimeric end oligonucleotide and the adaptor A, taking the functional chimeric end oligonucleotide and the adaptor B, respectively and gradually annealing to form double chains, then respectively adding Tn5 transposase for incubation to obtain a transposome A and a transposome B, mixing the transposome A and the transposome B, then adding an RNA/DNA hybrid product, and fragmenting in a buffer;
preparing a single bud library and sequencing: the fragmented RNA was PCR amplified and then purified to obtain the library-out product, which was quantified using a Qubit 2.0.
The sequence of the oligo-dTGVN primer is 5 '-AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3'.
In the reverse transcription reaction system, the sequence of the template switching oligonucleotide is
5’-AAGCAGTGGGTATCAACGCAGAGTACATrGrG+G-3’。
In the RNA reverse transcription step, the reverse transcription PCR reaction program is as follows: at 42 ℃ for 90 min; at 50 ℃ for 2 min; 42 ℃, 2min and 70 ℃, 15 min.
The sequence of the functional chimeric terminal oligonucleotide is 5-CTGCTCTTATACACATCT-3, the sequence of the joint A is 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', and the sequence of the joint B is 5 ' -GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3.
In the Tn5 transposition complex assembly and purification steps, the buffer included 10mM Tris, 5mM MgCl 210% N, N-dimethylformamide, 9% PEG8000, 0.85mM ATP.
In the RNA extraction step, Trizol extracting solution is adopted to extract total RNA from a single flower bud, chloroform is used for centrifugal separation, isopropanol is used for centrifugal separation to enable the RNA to be precipitated on the tube wall and the bottom, ethanol is used for washing, then air drying is carried out to be transparent, and double distilled water subjected to high-temperature sterilization is added to dissolve the RNA for later use.
The specific steps of PCR amplification of the fragmented RNA are as follows: 100units of Superscript II and 1x Q5High-Fidelity Master Mix were added to the fragmented RNA products at 42 ℃ for 15 min; 70 degrees and 15 min; then adding 5 mu L N5XX and 5 mu L N7XX to perform PCR amplification, wherein the reaction procedure is 72 ℃ and 15 min; 30s at 98 ℃; 10 cycles of 98 deg.C for 20s, 60 deg.C for 20s, and 72 deg.C for 2 min; 72 ℃ for 5 min.
After PCR amplification of fragmented RNA was complete, VAHTS DNA Clean Beads were used as per 1:1, purifying.
The present invention is further described in detail below by using micro RNA library construction of litchi chinensis Sonn or single flower bud of Ardisia oblonga.
Referring to fig. 2, a method for single bud micro RNA library construction includes the following steps:
101. pTXB1-Tn5 plasmid was purchased.
102. The pTXB1-Tn5 plasmid was transformed into expression strain Transetta (DE3) competent cells, plated on ampicillin (50mg/L) resistant LB medium overnight, and a single clone was selected and cultured in 10mL of LB liquid medium (ampicillin-resistant Amp) at 37 ℃ overnight with shaking at 250 rpm.
103. 10. mu.L of the stored Tn5 strain (DE3 strain) was pipetted into 10mL of LB liquid containing 50mg/L of ampicillin at 37 ℃ and 220rpm, which required approximately 18 hours.
104. The inoculum from step 103 was transferred (preferably at a ratio of 1:50 or 1: 100) to 200mL LB medium, ampicillin (50mg/L) was added, the culture was continued at 37 ℃ and 250rpm, and the OD value of the inoculum was measured intermittently so that OD600 became 0.9 (transfer at a ratio of 1:100, approximately 6 h). 1.5ml of the suspension was taken as a whole cell control before IPTG induction and stored at-20 ℃ as shown in S1 of FIG. 1.
105. Then the bacterial liquid in step 104 is cooled to 10 ℃, and 0.25mM isopropyl-beta-D-thiogalactoside is added for induction expression. After further growth at 220rpm at 23 ℃ for 4h, the OD600 reached 3.0, yielding 3-4gr cells. 1.5ml of the suspension was used as a whole cell control after IPTG induction and stored at-20 ℃ as shown in S2 of FIG. 1.
106. And (3) collecting the bacterial liquid obtained in the step (105) into a 50mL centrifuge tube, centrifuging at 12000r/min at 4 ℃ for 15min, removing supernatant, and then freezing the bacterial liquid precipitate at-80 ℃ overnight.
107. The pellet of step 106 was dissolved on ice, then added to 16mL of HEGX lysate and 640. mu.L of protease inhibitor and gently blown to resuspend the pellet, avoiding air bubbles. The HEGX lysate comprises 20mM HEPES-KOH (pH 7.2), 0.8M NaCl, 1mM EDTA, 10% glycerol, and 0.2% Triton X-100.
108. And (4) carrying out ultrasonic crushing on ice by using an ultrasonic crusher until the bacterial liquid is semitransparent. The power of the sonicator was set to 20%, sonicated for 9s, stopped for 9s, the frequency and intensity were adjusted to minimize bubble/foam formation. 1mL of the supernatant was aspirated for SDS-PAGE analysis-detection of the efficiency of cell disruption, as shown in S3 of FIG. 1.
109. The lysate from step 108 is centrifuged in a centrifuge at 12,000rpm for 30 minutes at 4 ℃.
110. The supernatant from step 109 was collected, and the precipitate was discarded, followed by dropwise addition of 420. mu.L of a 10% neutralized polyethyleneimine solution. The precipitate was then removed by centrifugation at 12,000rpm for 10 minutes at 4 ℃. Introduction of the polyethyleneimine precipitation step can effectively reduce contamination of reads of E.coli in the library. mu.L of the supernatant was retained for SDS-PAGE analysis-crude cell extract as shown in S4 of FIG. 1.
111. Preparing chromatographic columns, filling a cotton pad, washing the columns by using HEGX solution containing protease inhibitor, then thoroughly mixing column materials, adding 2mL of column material into each chromatographic column, standing until the column materials are settled, adding 4-5mL of HEGX solution, and passing through the column until a little liquid remains in the chromatographic column.
112. The chromatographic column bottom cap was sealed, then 8mL of the supernatant from step 110 was added to each column and the lid was closed.
113. Rotate on the rotator at 5rpm for 25 min.
114. Taking down the chromatographic column, standing until layering, opening the top cover and then the bottom cap, collecting the effluent, and storing at 4 ℃. 10 μ L of the effluent was removed for SDS-PAGE analysis, as shown in S5 of FIG. 1.
115. The column was washed with 30mL of HEGX solution (. gtoreq.20 bed volume of column buffer) to completely remove free protein and the wash was collected. 10 μ L of the wash was removed for SDS-PAGE analysis, as shown in S6 of FIG. 1.
116. Addition of mercaptan: the bottom lid was closed and 5mL of HEGX solution (containing protease inhibitor) containing 100mM DTT was added to the bottom of the bed.
117. On-column lysis: the column of step 116 was kept closed at 4 ℃ and 5rpm for 36-48 hours to effect Tn5 cleavage from the intron.
118. And (3) taking the chromatographic column in the step 117 from the rotator, standing for 5-10min, and collecting the liquid containing the target protein after cutting the chromatographic column to obtain a primary non-concentrated solution of Tn5 protein. 10 μ L of the supernatant was retained for SDS-PAGE analysis as shown in S8 lane of FIG. 1.
119. The column was eluted once with 2ml of a solution of LHEGX (run-off after standing for 10 min) and the residual Tn5 protein was collected. mu.L of the eluate was removed for SDS-PAGE analysis, as shown in S9 lane of FIG. 1.
120. Dialyzing with ultrafiltration concentration column. mu.L of the waste filtrate was retained for SDS-PAGE analysis-Tn 5 dialysis efficiency test, as shown in FIG. 1, lane S11.
121. When 200-.
122. The inside of the dialysis column was slowly purged with a gun and the concentrated Tn5 protein was aspirated. 10 μ L of the suspension was taken for SDS-PAGE analysis, and FIG. 1, lane S10.
123. 1.1V of 100% glycerol was added to each tube for storage.
124. The Tn5 protein concentration was determined using a Qubit.
Step 20, RNA extraction step:
201. putting the single flower buds of litchi/Ardisia obovata Linn into a 1.5mL centrifuge tube, thoroughly grinding with a pestle under the low-temperature condition created by liquid nitrogen, adding 300 mu L Trizol extracting solution, shaking and uniformly mixing the mixture upside down, and standing the mixture for 3-5 minutes at room temperature.
202. Adding 100 mu L of chloroform into the centrifuge tube, shaking vigorously, standing for 3-5 minutes at room temperature, and centrifuging for 15 minutes in a refrigerated centrifuge with the rotation speed of 12000rpm and precooling to 4 ℃.
203. After centrifugation, the supernatant is absorbed and transferred into a new 1.5mL centrifuge tube, isopropanol with the same volume is added, the mixture is turned upside down and mixed softly and evenly, the mixture is kept stand for 10 minutes at room temperature, the mixture is centrifuged for 10 minutes in a refrigerated centrifuge with the rotation speed of 12000rpm and precooled to 4 ℃, and RNA is precipitated on the tube wall and the bottom.
204. The supernatant was discarded, 1mL of 75% ethanol was added for washing, and the mixture was centrifuged at 7500rpm for 5 minutes in a refrigerated centrifuge cooled to 4 ℃ to remove 75% ethanol.
205. The above washing step was repeated and 75% ethanol was decanted.
206. The empty tube was centrifuged at 12000rpm for 3 minutes in a refrigerated centrifuge precooled to 4 ℃ to suck off excess alcohol, the alcohol was air-dried in a fume hood until it became transparent, and 10. mu.L of ddH2O sterilized at high temperature for 40 minutes was added to dissolve RNA.
207. 1 μ L was aspirated for RNA purity and concentration, and the sample concentration and OD ratio were recorded, with the results shown in Table 1.
Step 30, RNA reverse transcription step:
301. 2.3uL of RNA was added to a 0.2mL thin-walled PCR tube. mu.L of 10. mu.M oligo-dTGN primer and 1. mu.L dNTP mix were added. The sequence of the oligo-dTGN primer is as follows:
5’-AAGCAGTGGTATCAACGCAGAGTACT30VN-3’)
302. vortex rapidly and mix well, centrifuge at room temperature for 10 seconds at 700g, ice-bath immediately.
303. The samples were incubated at 72 ℃ for 3 minutes with an immediate ice bath.
304. 700g at room temperature for 10s, collect the tube bottom liquid and immediately ice bath for standby.
305. Preparing a reverse transcription reaction system: 100U SuperScript II reverse transcriptase (200U/. mu.L), 10U RNase inhibitor (40U/. mu.L), 1 XPRESTRAPT II first strand buffer, 5mM DTT, 1M betaine, 6mM MgCl21 μ M TSO. The TSO sequence is as follows: 5 '-AAGCAGTGGGTATCAACGCAGAGTACATRGRGG + G-3'.
306. The reverse transcription reaction system was added to the sample obtained in step 304, and the final reaction system was made to be 10. mu.L by filling with water. And (4) blowing and sucking up and down gently to mix uniformly, so as to avoid generating bubbles.
307. Centrifuging at room temperature of 700g for 10s, collecting liquid at the bottom of the tube, and placing the liquid in a PCR instrument for reverse transcription PCR reaction, wherein the PCR reaction procedure is as follows: 90min at 42 ℃; 50 ℃, 2min and 42 ℃, 2min (10 cycles); 70 ℃ for 15 min.
Step 40, Tn5 transposable Complex Assembly and purification steps
401. Respectively placing 50uL functional chimeric end (ME) oligonucleotide and 50uL Adaptor A in an enzyme-free 200uL PCR tube, mixing, and heating at 95 deg.C for 3 min; 3min at 70 ℃; 44 cycles [70 ℃ -25 ℃, 1 ℃ reduction per cycle finally to 25 ℃, 30s of each temperature operation, then to return to normal temperature, then can be used directly, or stored at-20 ℃. The same method is adopted to anneal ME and Adaptor B step by step to form double chains.
The sequence of the functional chimeric end oligonucleotide is 5-CTGCTCTTATACACATCT-3.
The sequence of the linker Adaptor a is 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'.
The sequence of the linker adapter B is 5' -GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3.
402. 100ug of Tn5 transposase preserved with glycerol was thoroughly mixed with ME-Adaptor A (20. mu.M) or ME-Adaptor B (20. mu.M), respectively, annealed in the above step 401, and incubated at 30 ℃ for 1 hour to obtain transposome A and transposome B.
403. The two turret bodies obtained in step 402 are mixed together thoroughly.
404. Excess linker sequence in the transposer body from step 403 above was removed using an Amicon Ultra-0.5mL centrifugal filter, prepared for fragmentation or stored at-20 ℃.
405. For the RNA/DNA hybrid product obtained by reverse transcription of RNA in step 30, 0.006uL of the Tn5 transposition complex of step 404 was added in the presence of 10mM Tris-Cl (pH 7.6), 5mM MgCl2Fragmentation was performed in a buffer of 10% N, N-dimethylformamide, 9% PEG8000, 0.85mM ATP and incubation at 55 ℃ for 30 min.
Step 50, preparation and sequencing of single bud library
501. To the above-mentioned fragmentation product, 100units of Superscript II and 1x Q5High-Fidelity Master Mix were added and reacted at 42 ℃ for 15min, followed by reaction at 70 ℃ for 15min to inactivate the Superscript II. The specific reaction procedure is as follows: 42 ℃ for 15 min; 70 ℃ and 15 min.
502. Indext common primers (5. mu. L N5XX + 5. mu. L N7XX) were added for PCR amplification with the sequence of 72 ℃ for 15min, 98 ℃ for 30s, 10 cycles [98 ℃ for 20s, 60 ℃ for 20s, 72 ℃ for 2min ], 72 ℃ for 5 min.
503. Firstly, 5-8 cycles of reaction are carried out by a PCR instrument, then 10uL of reaction products are sucked for qRT-PCR, the CT value of each library is recorded, and then the cycle number is set according to the CT value (generally 1-2 cycles are added on the basis of the CT value) for PCR amplification of the residual reaction products.
504. The library obtained after completion of PCR was purified using VAHTS DNA Clean Beads as per 1:1 ratio for purification.
505. The products from the library were obtained, quantified using a Qubit 2.0, and submitted to the assay according to the sequencing requirements, with the results shown in table 1.
TABLE 1 Single flower bud RNA and information sheet related to library concentration and transcriptome data
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A single bud micro RNA library building method is characterized by comprising the following steps:
pTXB1-Tn5 purification step: transformation of the pTXB1-Tn5 plasmid into competent E.coli Transetta DE 3; inoculating a single colony into an LB culture medium added with antibiotics, and culturing until OD600 is 0.9; adding 0.25mM isopropyl-beta-D-thiogalactoside for induction expression until OD600 is 3.0, and centrifuging the bacteria liquid after induction expression to prepare thalli; adding HEGX lysate and protease inhibitor, and cracking to release protein; centrifuging the cracked product, taking supernatant, adding a PEI solution, centrifuging to remove precipitates, and performing chromatography on the obtained supernatant; then cracking the chromatographic column to realize the cutting of the pTXB1-Tn5 plasmid from the intron; collecting liquid containing target protein to obtain a Tn5 protein unconcentrated solution; then dialyzing by using an ultrafiltration concentration column to obtain Tn5 transposase;
RNA extraction step: extracting RNA from a single bud of a plant;
RNA reverse transcription step: adding oligo-dTGN primer and dNTP mixture into the solution dissolved with RNA to prepare RNA sample solution for later use; preparing a reverse transcription reaction system, wherein the reverse transcription reaction system comprises SuperScript II reverse transcriptase, an RNase inhibitor, a SuperScript II first chain buffer solution, dithiothreitol, betaine, magnesium chloride and template switching oligonucleotide; adding the RNA sample solution into a reverse transcription reaction system, and operating PCR reaction to obtain an RNA/DNA hybrid product;
tn5 transposition complex assembly and purification steps: taking functional chimeric terminal oligonucleotide and adaptor A, taking functional chimeric terminal oligonucleotide and adaptor B to anneal step by step respectively to form double chains, then adding Tn5 transposase respectively to incubate to obtain transposome A and transposome B, mixing transposome A and transposome B, adding RNA/DNA hybrid product, and fragmenting in buffer;
preparing a single bud library and sequencing: the fragmented RNA was PCR amplified and then purified to obtain the library-out product, which was quantified using a Qubit 2.0.
2. The single bud microrna library construction method of claim 1, wherein: the sequence of the oligo-dTGVN primer is 5 '-AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3'.
3. The single bud microrna banking method of claim 1 wherein: in the reverse transcription reaction system, the sequence of the template switching oligonucleotide is 5 '-AAGCAGTGGGTATCAACGCAGTACATRGRGRGRGG + G-3'.
4. The single bud microrna library construction method of claim 1, wherein: in the RNA reverse transcription step, the reverse transcription PCR reaction program is as follows: at 42 ℃ for 90 min; at 50 ℃ for 2 min; 42 ℃ for 2min and 70 ℃ for 15 min.
5. The single bud microrna library construction method of claim 1, wherein: the sequence of the functional chimeric terminal oligonucleotide is 5-CTGCTCTTATACACATCT-3, the sequence of the joint A is 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3', and the sequence of the joint B is 5 ' -GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3.
6. The single bud microrna library construction method of claim 1, wherein: in the Tn5 transposition complex assembling and purifying step, the buffer comprises 10mM tris, 5mM MgCl210% N, N-dimethylformamide, 9% PEG8000, 0.85mM ATP.
7. The single bud microrna library construction method of claim 1, wherein: in the RNA extraction step, Trizol extracting solution is adopted to extract total RNA from a single flower bud, chloroform is used for centrifugal separation, isopropanol is used for centrifugal separation to enable the RNA to be precipitated on the tube wall and the bottom, ethanol is used for washing, then air drying is carried out to be transparent, and double distilled water subjected to high-temperature sterilization is added to dissolve the RNA for later use.
8. The single flower bud microRNA library construction method of claim 1, wherein the step of PCR amplifying the fragmented RNA is: 100units of Superscript II and 1x Q5High-Fidelity Master Mix were added to the fragmented RNA products at 42 ℃ for 15 min; 70 degrees and 15 min; then adding 5 mu L N5XX and 5 mu L N7XX to perform PCR amplification, wherein the reaction procedure is 72 ℃ and 15 min; 30s at 98 ℃; 10 cycles of 98 deg.C for 20s, 60 deg.C for 20s, and 72 deg.C for 2 min; 72 ℃ for 5 min.
9. The single bud microrna library construction method of claim 8, wherein: after PCR amplification was complete, the PCR was performed using VAHTS DNA Clean Beads as described in 1:1, purifying.
10. The method for establishing the library of the single flower bud micro RNA of claim 1, wherein the single flower bud is taken as a litchi chinensis Sonn or a single flower bud of a Redbud.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002569A (en) * | 2015-07-15 | 2015-10-28 | 北京诺禾致源生物信息科技有限公司 | Transcriptome library and construction method thereof |
| CN107580632A (en) * | 2015-04-23 | 2018-01-12 | 赛卢拉研究公司 | Method and composition for the amplification of full transcript profile |
| WO2018103025A1 (en) * | 2016-12-07 | 2018-06-14 | 深圳华大智造科技有限公司 | Method for constructing single cell sequencing library and use thereof |
| CN109930206A (en) * | 2017-12-18 | 2019-06-25 | 深圳华大生命科学研究院 | Detection kit is sequenced in micro blood platelet RNA based on BGISEQ-500 |
| CN112176421A (en) * | 2020-09-22 | 2021-01-05 | 上海英基生物科技有限公司 | RNA library building method |
| CN112251422A (en) * | 2020-10-21 | 2021-01-22 | 华中农业大学 | Transposase complex containing unique molecular tag sequence and application thereof |
| CN113061648A (en) * | 2021-03-24 | 2021-07-02 | 中山大学 | Method for constructing micro sample m6A modification detection library by aid of Tn5 transposase and application of method |
| CN113186174A (en) * | 2021-04-23 | 2021-07-30 | 江南大学 | Preparation and application of Tn5 mutant enzyme |
-
2022
- 2022-03-07 CN CN202210225713.2A patent/CN114645074A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107580632A (en) * | 2015-04-23 | 2018-01-12 | 赛卢拉研究公司 | Method and composition for the amplification of full transcript profile |
| CN105002569A (en) * | 2015-07-15 | 2015-10-28 | 北京诺禾致源生物信息科技有限公司 | Transcriptome library and construction method thereof |
| WO2018103025A1 (en) * | 2016-12-07 | 2018-06-14 | 深圳华大智造科技有限公司 | Method for constructing single cell sequencing library and use thereof |
| CN109930206A (en) * | 2017-12-18 | 2019-06-25 | 深圳华大生命科学研究院 | Detection kit is sequenced in micro blood platelet RNA based on BGISEQ-500 |
| CN112176421A (en) * | 2020-09-22 | 2021-01-05 | 上海英基生物科技有限公司 | RNA library building method |
| CN112251422A (en) * | 2020-10-21 | 2021-01-22 | 华中农业大学 | Transposase complex containing unique molecular tag sequence and application thereof |
| CN113061648A (en) * | 2021-03-24 | 2021-07-02 | 中山大学 | Method for constructing micro sample m6A modification detection library by aid of Tn5 transposase and application of method |
| CN113186174A (en) * | 2021-04-23 | 2021-07-30 | 江南大学 | Preparation and application of Tn5 mutant enzyme |
Non-Patent Citations (2)
| Title |
|---|
| 吴璇等: "荷花花蕾cDNA文库构建及质量评价", 《亚热带植物科学》, no. 01, pages 48 - 52 * |
| 许昊等: "基于工程化转座酶的少量细胞RNA-seq文库构建的研究", 《第三军医大学学报》, no. 09, pages 1 * |
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