CN117187347A - mRNA-cDNA enrichment method applied to bacterial single cell sequencing - Google Patents

Abstract

The invention discloses an mRNA-cDNA enrichment method applied to bacterial single cell sequencing, belonging to the technical field of biology. The method comprises the following steps: 1) Preparing a bacterial total cDNA containing a 5' specific sequence; 2) Template conversion is carried out on the total cDNA; 3) RNaseH digests RNA strands hybridized to cDNA; 4) Molecular hybridization is carried out on single-stranded cDNA, r-cDNA probes and 3' -end biotin-labeled primers, and streptavidin magnetic beads are utilized to remove biotin-containing molecules, so that enriched mRNA-cDNA is obtained. The two ends of the enriched mRNA-cDNA are known sequences, and DNA library construction can be completed through the steps of PCR amplification, end repair, linker connection and the like. The invention only needs the user to customize the probe sequence according to the needs, and has the advantages of simple use, rapidness, high efficiency, low cost, no special instrument consumable material and the like.

Description

mRNA-cDNA enrichment method applied to bacterial single cell sequencing

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an mRNA-cDNA enrichment method applied to bacterial single cell sequencing.

Background

Transcriptome sequencing is a method for quickly knowing all transcript information of bacterial cells in a certain state, and is an important technical means in biomedical research. Unlike eukaryotes, bacterial mRNAs do not contain a 3-terminal poly (A) structure, and thus mRNAs cannot be reverse transcribed specifically by oligo (dT) to obtain mRNA information. On the other hand, the ratio of the mRNA of bacteria to the total RNA is extremely low, for example, the mRNA in the escherichia coli in logarithmic growth phase only accounts for about 5-8% of the total RNA, so that the enrichment of the mRNA is very important for constructing a bacterial transcriptome library. Currently, to increase the mRNA detection rate of bacterial cell transcriptome sequencing, the main technical methods are: 1) Adding rRNA probe to the total RNA, hybridizing with rRNA to form DNA-RNA hybrid strand, and degrading rRNA probe with RNaseH (e.g.: n407 product of Vazyme); 2) Adding a biotin-labeled rRNA probe into the total RNA, hybridizing the rRNA probe with the rRNA to form a DNA-RNA hybrid chain, and removing the hybrid chain (such as an AM1905 product of Thermo); 3) Other techniques, etc. (unusual). These methods operate at an excessive level of RNA increasing the extent of RNA degradation; on the other hand, for techniques such as single cell transcriptome sequencing based on bacterial in situ markers (1, 2), such methods are difficult to apply or do not perform well because the reaction is performed in bacteria.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides an enrichment method of bacterial mRNA-cDNA and application of the enrichment method in constructing a bacterial single-cell transcriptome sequencing library or bacterial transcriptome sequencing, including construction of a single-cell transcriptome sequencing library based on bacterial in-situ labeling and a conventional bacterial transcriptome sequencing library. The enrichment method can efficiently and rapidly enrich cDNA (m-cDNA, rRNA-derived cDNA written r-cDNA) derived from mRNA from a single-strand cDNA library constructed by bacterial single-cell sequencing, and construct a single-cell transcriptome sequencing library on the basis.

The aim of the invention is achieved by the following technical scheme:

a method for enriching mRNA-cDNA of a bacterium, comprising the steps of:

(1) Reverse transcription of bacterial total RNA using random reverse transcription primers containing 5' terminal specific sequences to obtain total cDNA. This step can be directed against in situ labelled bacterial single cells, or can be extended for application to bacterial total RNA extracted in vitro. The addition of specific sequences in random reverse transcription primers provides specific primer binding targets for subsequent library PCR amplification.

The sequence of the reverse transcription primer is as follows: 5' -X _(18-25) N ₆ -3'. Wherein N is ATCG random one, and the primer is synthesized randomly; x: during synthesis, a user specifically identifies the amplified primer sequence as one of ATCG according to the subsequent library PCR amplification primer sequence, and the GC content is required to be between 40 and 60 percent. For example: 5'-CTTCGACCTCCTACGCCAGANNNNNN-3' (SEQ ID NO. 1).

Note that: aiming at the construction of a bacterial single cell transcriptome sequencing library, the need of single cell labeling on cells can be properly reduced to about 10-15's of the length of the "X" of the reverse transcription primer, and the 5' -most end is subjected to phosphorylation modification so as to carry out the connection of specific DNA fragments at the 5' -end of the reverse transcription primer. In summary, it is ensured that the 5' -end of the random sequence 5' -NNNNNN-3' in the reverse transcription primer is added with a certain specific sequence (the GC content is between 40 and 60 percent), and the addition of the specific sequence provides a specific primer binding target for the subsequent library PCR amplification.

(2) Template conversion is carried out on the cDNA obtained in the step (1): the cDNA was again "reverse transcribed" using the feature that the cDNA end was the CCC sequence, using a template switching primer (TSO) containing a 5' end specific sequence. The 3' end of the cDNA converted by the template is added with a specific sequence, and the addition of the specific sequence provides a specific primer binding target for the subsequent PCR amplification of the library.

The sequence of the TSO primer is as follows: 5' -X _(25-30) rGrG+G-3'. Wherein "rG" is ribonucleic guanines; "+G" is locked nucleic acid guanine, which is specifically identified by the user as one of ATCG based on the sequence of the primers amplified by the subsequent library PCR during X synthesis, and requires GC content of 40-60%. For example: 5 '-AAGCAGTGGTATCACGCACGCAGGTACATrGrG+G-3' (SEQ ID NO. 2).

(3) The cDNA subjected to template conversion is a DNA-RNA hybrid strand, and RNA in the hybrid strand is digested with RNaseH to expose single-stranded cDNA.

(4) Molecular hybridization is carried out on the single-stranded cDNA, the r-cDNA probe library and the 3' -end biotin-marked primer, and the streptavidin magnetic beads are combined with biotin to remove the biotin-marked primer and the hybridized molecular complex thereof, thus obtaining the enriched mRNA-cDNA.

Said rThe sequence of the cDNA probe is: 5' -X _(20-30) Y _(10-15) Z _(20-30) -3'. X, Y, Z is an ATCG, X, Y requires GC content of 40-60% for design. X is X _(20-30) For hybridization with the biotin-labeled primer, so that its sequence is reverse-complementary to the biotin-labeled primer. Y is Y _(10-15) For separating X from Z to form a non-hybridizing region. Z is Z _(20-30) For hybridization with r-cDNA, so that its sequence is reverse complementary to r-cDNA, or identical to rRNA sequence. For example, E.coli 16S rRNA sequence 5'-AAATTGAAGAGTTTGATCATGGCTC-3', the synthesized probe was 5'-TTCAAGACTAGGGTGCTTGCGGCTCCGAGTAAATTGAAGAGTTTGATCATGGCTC-3' (SEQ ID NO. 3).

The sequence of the biotin-labeled primer can be identical to that of X of the above-mentioned r-cDNA probe _(20-30) Hybridization is carried out, the sequence of which hybridizes with X _(20-30) Reverse complement, for example: 5'-GCAAGCACCCTAGTCTTGAA-biotin-3' (SEQ ID NO. 4).

In the step (1), the method for obtaining total cDNA by reverse transcription of total RNA of bacteria is as follows:

1) A method for total RNA in vitro comprising the steps of: extracting total RNA of the escherichia coli by a phenol-chloroform method or a commercial kit method, and carrying out reverse transcription on the total RNA by using a reverse transcription primer to synthesize total cDNA. Reverse transcriptase should be selected from products with no (or low) RNaseH activity. The total cDNA was purified and enriched using DNA purification magnetic beads.

2) A method for labeling bacterial single cells in situ comprising the steps of: referring to the method (1, 2) for in-situ labeling single cells of bacteria, the bacterial cells are subjected to operations such as fixation, permeation, chromosomal DNA digestion and the like. And then carrying out random reverse transcription in bacterial cells by using reverse transcription primers to synthesize total cDNA. The optional steps are as follows: ligation of one or more DNA fragments at the 5' end of the reverse transcription primer is performed to label single cells (1, 2), or other modification procedures.

The mRNA-cDNA obtained by the above method has known sequences at both ends, respectively, from the specific sequence in the reverse transcription primer and the TSO primer. Library amplification primers can be designed accordingly. For example: 5'-CTTCGACCTCCTACGCCAGA-3' (SEQ ID NO. 5) and 5'-CAGTGGTATCAACGCAGAGTAC-3' (SEQ ID NO. 6), the mRNA-cDNA was amplified specifically by PCR using this primer. And then, a commercial DNA library amplification kit is selected according to the requirement, and the PCR products are subjected to end repair, joint connection, DNA purification and the like to obtain a bacterial transcriptome sequencing library. The constructed library can be directly used for second generation sequencing.

The enrichment method of the bacterial mRNA-cDNA has application in constructing a bacterial transcriptome sequencing library or bacterial transcriptome sequencing, and comprises construction and sequencing of a single-cell transcriptome sequencing library based on bacterial in-situ markers and a conventional bacterial transcriptome sequencing library.

Compared with the prior art, the invention has the advantages and beneficial effects that:

the invention provides an effective mRNA enrichment method aiming at cDNA level operation, which only needs a user to customize a probe sequence according to the needs, is convenient and economic to use, and does not need special instrument consumables and the like.

The invention solves the problem that the single cell transcriptome sequencing of the bacteria in-situ mark can not obtain high mRNA information.

Drawings

FIG. 1 is a schematic flow chart of the method of the invention, wherein first, a random reverse transcription primer containing a 5' end specific sequence is utilized to carry out reverse transcription on total RNA of bacteria to obtain total cDNA; purifying the total cDNA and performing template conversion on the cDNA; digesting the hybridized RNA strand in the strand cDNA by RNaseH; targeting hybridization of r-cDNA using probes designed for rRNA and hybridization with biotin-labeled universal primers; removing the biotin-labeled primer (and the hybridized molecular complex thereof) by using streptavidin magnetic beads to obtain enriched m-cDNA; designing a specific primer amplification library by utilizing specific sequences at two ends of the cDNA; and (3) repairing the tail end and connecting the joints of the library to complete library construction.

FIG. 2 shows the proportion of mRNA source reads in transcriptome libraries constructed by different methods.

FIG. 3 is a correlation of transcriptome libraries constructed by different methods.

FIG. 4 shows the mRNA and rRNA ratios used in the various species of the method of the invention.

Detailed Description

The following is a specific example. The invention is further illustrated with the understanding that these examples are intended to be illustrative of the invention and are not intended to limit the scope of the invention as claimed.

Example 1 construction of E.coli Single cell transcriptome library based on bacterial in situ labelling

Briefly described: in this example, the total RNA was reverse transcribed and further modified in bacteria against E.coli in exponential growth phase, and the total cDNA from a population of bacteria was purified and library constructed according to the method of the invention.

1) Pretreatment of bacteria

1.1 preparing a fungus sample; 5mL of log-phase wild MG1655 cells were centrifuged at 5525g at 4℃for 2min, and the medium was discarded. The cells were resuspended with 5mL of pre-chilled 4% formaldehyde and placed on a flip-flop at 4℃for 16h or overnight.

1.2 taking 1mL of immobilized thallus 5000g, centrifuging at 4 ℃ for 5min, and removing supernatant. The cells were resuspended in 1mL Washing Buffer (100 mM Tris-HCl pH7, 0.02U/. Mu.L SUPERAse-In RNase Inhibitor), centrifuged at 5000g at 4℃for 5min and the supernatant removed.

1.3 the cells were resuspended in 250. Mu. L Permeabilization Buffer (0.04% Tween-20in PBS) and ice-bathed for 3min; 1mL of ice-bath PBS-RI (0.01U/. Mu.L of SUPERase In RNase Inhibitor was added to PBS) was added, centrifuged at 5000g for 5min at 4℃and the supernatant was discarded.

1.4 cells were resuspended in 250. Mu.L of Lysozyme Mix (250. Mu.g/mL of Lysozyme dissolved in TEL-RI:100mM Tris,pH 8.0;50mM EDTA;0.1U/. Mu.L SUPERase In RNase Inhibitor) and incubated for 15min in a metal bath at 37 ℃.

1.5 immediately add 1mL of ice-bath PBS/RI, centrifuge at 5000g for 5min at 4deg.C, discard supernatant. Repeating once.

1.6 cells were resuspended with 40. Mu.L DNaseI-RI buffer (4.4. Mu.L 10 Xreaction buffer, 0.2. Mu. L SUPERase In RNase inhibitor, 35.4. Mu. L H) ₂ O) was added with 4. Mu.L of DnaseI (from AMPD1, millipore Sigma) and incubated at room temperature for 30min.

1.7 adding 4 mu L Stop Solution, placing in a metal bath at 50 ℃ for incubation for 10min, and turning over and mixing once every 1 min. The supernatant was removed by centrifugation at 7000g for 10min at 4 ℃.

1.8 the cells were resuspended in 500. Mu.L of PBS-RI, centrifuged at 7000g for 10min at 4℃and the supernatant removed. This step is repeated once more.

2) Synthesis of Total cDNA

2.1 primer design: the primers designed for use in this example are shown in tables 1-3 and include 96Round1 RT primers,96 Round2 ligation primers, round3 ligation primers, etc.

2.2 primer pretreatment: the 96 kinds of Round2 ligation primers (100. Mu.M) were each used in 31.1. Mu.L, 28.5. Mu.L SB83 (100. Mu.M) was added thereto, and 21.4. Mu. L H was added thereto ₂ O, respectively split charging 2.24 mu L into eight-joint tubes for standby after uniformly mixing. The 96 kinds of Round3 ligation primers (70. Mu.M) were prepared by taking 63.2. Mu.L each, adding 58. Mu.L SB80 (70. Mu.M), mixing well, and then sub-packaging 3.49. Mu.L each in an eight-way tube for use. 50. Mu.L of SB84 (400. Mu.M) and 80. Mu.L of SB81 (400. Mu.M) were taken at 94℃for 3min and then slowly reduced to 25℃at-0.1℃to form an intramolecular hairpin structure. Preparing Round 2blocking mix: 37.5. Mu.L 400. Mu.M SB84, 37.5. Mu.L 400. Mu.M SB85, 25. Mu.L 10×T4 livegasbuffer, 150. Mu. L H ₂ O. Preparing a Round 3blocking mix:72 μL400 μM SB81, 72 μL400 μM SB82, 120 μL 10×T4 ligase buffer,336 μ L H ₂ O，600μL 0.5M EDTA。

2.3 taking 3X 10 ⁷ The above bacteria (1.8) were added to 220. Mu.L of 5 XRT buffer, 22. Mu.L of dNTPs, 11. Mu. L SUPERase In RNase Inhibitor, 22. Mu. L Maxima H Minus Reverse Transcriptase (EP 0753, thermo Scientific), 132. Mu.L of 50% PEG8000, with H ₂ O is added to 880 mu L, and the mixture is blown and evenly mixed.

2.4 2. Mu.L of Round1 RT primers were added for each 8. Mu.L of mix (2.3) above, 96 total. And (3) placing the mixture in a PCR instrument for reaction: preserving at 50deg.C for 10min, 8deg.C for 12s, 15deg.C for 45s, 20deg.C for 45s, 30deg.C for 30s, 42deg.C for 6min, 50deg.C for 16min, 4deg.C.

2.5 the reaction solution of all wells in 96-well plates was collected in a 1.5mL centrifuge tube (ice-operated), 75. Mu.L of 0.5% Tween-20 (final concentration 0.04%) was added, ice-bath was carried out for 3min, and centrifuged at 7000g at 4℃for 10min, and the supernatant was removed. Then, 400. Mu.L of PBS-RI was used for resuspension, 32. Mu.L of 0.5% Tween-20 was added, and the mixture was centrifuged at 7000g at 4℃for 10 minutes to remove the supernatant.

2.6 the cells were resuspended in 500. Mu.L of 1 XT 4 library buffer and the following reagents were added: 107.5. Mu.L PEG8000, 37.5. Mu.L 10 XT 4 ligase buffer, 16.7. Mu. L SUPERase In RNase Inhibitor, 5.6. Mu.L BSA (enzyme digestion kit), and 27.9. Mu. L T4. Mu.4 ligase (M0202L, NEB), total volume 695.2. Mu.L, were well mixed (vigorously vortexed). Split 5.76 μl into pretreated middle Round 2primers mix and react at 37deg.C for 45min. mu.L of Round 2blocking mix was added, and the reaction was continued at 37℃for 45min.

2.7 the reaction solutions in the 96-well PCR plates were collected together. Adding 89 mu L H ₂ O, 26. Mu.L PEG8000, 46. Mu.L 10×T4 ligase buffer, and 12.65. Mu. L T4 ligase were blown and mixed. Split 8.51 μl into pretreated Round 3primers mix and react at 37deg.C for 45min. mu.L of Round 3blocking mix was added, and the reaction was continued at 37℃for 45min.

2.8 taking appropriate amount of cells, adding into 50. Mu.L of lysis buffer (50mM Tris pH8.0, 25mM EDTA,200mM NaCl,0.5%Triton X-100), adding 5. Mu.L of protease K, incubating in a metal bath at 55deg.C for 60min, and mixing every 2 min. Purification with 2 Xratio of Northenpran VAHTSDNA Clean Beads and finally cDNA was dissolved in 12. Mu. L H ₂ O。

The 5' end of the obtained cDNA contains the sequence: 5'-AGAATACACGACGCTCTTCCGATCT-3', and the library amplification primer R3 is designed according to the following steps: 5'-ATACACGACGCTCTTCCGATC-3'.

3) Template conversion

3.1 template transformation with one strand cDNA. The following reaction liquid is prepared: mu.L of cDNA, 4. Mu.L of 5 XRT buffer, 1. Mu.L of dNTPs, 0.5. Mu. L SUPERase In RNase Inhibitor, 0.5. Mu. L Maxima H Minus Reverse Transcriptase, 2. Mu.L of TSO Primer (Table 1) were blow mixed.

3.2 reaction on PCR instrument as follows: preserving at 25deg.C for 30min, 42deg.C for 90min, 85deg.C for 5min, and 4deg.C.

4) RNaseH digestion

4.1 adding 1. Mu.L RNaseH into the template transfer product, mixing well, and reacting at 37 ℃ for 30min.

4.2 was purified with 2 Xratio of Norpran VAHTSDNA Clean Beads and dissolved in 12.5. Mu. L H ₂ O。

5) Biotin probe hybridization

5.1 preparation of probes: each probe designed for synthesis (Table 3) was formulated at 10. Mu.M, and all probes were mixed together in a 1:1 ratio as probes (libraries).

5.2 to the 12.5. Mu.L digested product was added 5. Mu.L of probe, 2.5. Mu.L of 10 Xhybridization buffer (Tris-HCl pH8.0100mM, naCl 500mM,EDTA pH8.0 10mM), 5. Mu.L of universal primer (10. Mu.M, table 1).

5.3, denaturation at 95℃for 2min, slow annealing to 25℃at 0.1℃per second, and incubation at 37℃for 30min.

6) Streptavidin removal of biotin

6.1 magnetic streptavidin beads (22307, beaver) ^TM ) 20. Mu.L, 1mL of 1 XB&W buffer (Tris-HCl pH7.510mM, EDTA 1mM,NaCl 1M,Tween20 0.05%) was washed 2 times with 25. Mu.L of 2 XB&W buffer resuspended S-beads.

6.2 to 25. Mu.L cDNA-biotin hybridization product 25. Mu. L S-beads were added, mixed and incubated at room temperature for 30min (mixing was reversed every 2 min).

6.3 magnetic separation of magnetic beads, supernatant, purification with 2 Xratio of Northenol VAHTSDNA Clean Beads, dissolution in 12.5. Mu. L H ₂ O。

7) Library amplification

7.1 to 12.5. Mu.L of the above-enriched m-cDNA were added 1.5. Mu.L of TSO-2, 1.5. Mu. L R3 (Table 1), 25. Mu.L of KAPA Hifi (KK 2602), 1. Mu.L of Sybr green (25X), 0.5. Mu.L of Mg ²⁺ (0.1M)，H ₂ O makes up 50. Mu.L.

7.2, after being evenly mixed, the mixture is placed in a fluorescent quantitative PCR instrument for PCR amplification according to the following procedure: 98℃45s,98℃15s,60℃30s,72℃60s. The number of PCR cycles is based on the amplification curve to logarithmic amplification period.

7.3 purification with 0.9 Xratio of Norflua VAHTSDNA Clean Beads, dissolved in 25. Mu. L H ₂ O。

8) Repairing terminal-linker junctions

8.1 end repair and linker ligation of the PCR products described above was performed with Norpraise ND 607.

8.2 purification of the linker ligation product with 0.6 Xratio of North praise VAHTSDNA Clean Beads. The product is the library finally constructed and can be used for Illumina second generation sequencing.

Note that: steps 5-6 were omitted as controls according to the procedure described above.

9) Sequencing result analysis

9.1m-cDNA enrichment Effect

In the transcriptome library constructed using the method of the present invention, the mRNA ratio increased from about 7.4% to about 77% in the control group. Transcriptome library construction was performed with Repran N407-NR603 on total RNA of E.coli MG1655 at log phase, with a ratio of reads to the corresponding region of mRNA of approximately 79.7% (FIG. 2). The result shows that the reads derived from mRNA in the escherichia coli single cell transcriptome library constructed by the invention is remarkably enriched, and has similar enrichment effect as that of a commercial Norflua N407-NR603 kit.

9.2m-cDNA enrichment does not affect the RNA-seq outcome

The above 5-6 steps are key steps for enriching m-cDNA. As shown in FIG. 3, the correlation between the control library constructed without the 5-6 steps and the library constructed by the method of the invention (case 1) was around 0.927, indicating that the m-cDNA enrichment step did not result in significant changes in the transcriptome library.

Example 2 construction of a single cell transcriptome library based on bacteria in situ labeled multiple bacteria (E.coli, P.aeruginosa, staphylococcus aureus, acinetobacter crescent)

The primers and library construction procedure designed in this example were the same as in example 1, except that 250. Mu.g/mL of Lysozyme was changed to the following reagents at the time of cell pretreatment: staphylococcus aureus was treated with 5. Mu.g/mL Lysostaphin, pseudomonas aeruginosa was treated with 250. Mu.g/mL Lysozyme, and Bacillus crescens was treated with 20. Mu.g/mL Lysozyme.

The method of the present invention works very well in a variety of gram negative and positive bacteria (FIG. 4). The mRNA proportion of about 8.5% of E.coli can be increased to about 81%; increasing the proportion of pseudomonas aeruginosa about 4.7% mRNA to about 76%; increasing the mRNA proportion of staphylococcus aureus to about 92% at about 9.7%; increasing the proportion of about 4.3% mRNA of the genus Acinetobacter to about 63%;

in summary, the present invention provides an efficient method of mRNA enrichment for cDNA level operations. The method only needs a user to customize the probe sequence according to the needs, is convenient and economical to use, and does not need special instrument consumables and the like.

Reference is made to:

1.A.Kuchina et al.,Microbial single-cell RNA sequencing by split-pool barcoding.Science 371,(Feb 19,

2021).

2.S.B.Blattman,W.Jiang,P.Oikonomou,S.Tavazoie,Prokaryotic single-cell RNA sequencing by in situ

combinatorial indexing.Nat Microbiol 5,1192(Oct,2020).

TABLE 1 primers according to the present invention

Table 2 96Round1 RT primers, round2 ligation priorities, round3 ligation priorities

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TABLE 3 Escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, and Currency Acinetobacter probe pool

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Claims (10)

1. A method for enriching mRNA-cDNA of a bacterium, comprising the steps of: the method comprises the following steps:

(1) Reverse transcription is carried out on the total RNA of the bacteria by using a random reverse transcription primer containing a 5' -end specific sequence to obtain total cDNA; the specific sequence in the reverse transcription primer provides a specific primer binding target for the subsequent library PCR amplification;

(2) Template conversion is carried out on the cDNA obtained in the step (1): performing reverse transcription again on the cDNA by using a TSO primer containing a 5' -end specific sequence; the specific sequence in the TSO primer provides a specific primer binding target for subsequent library PCR amplification;

(3) Digesting the template-converted cDNA with RNaseH to expose single-stranded cDNA;

(4) Carrying out molecular hybridization on single-stranded cDNA, r-cDNA probes and 3' -end biotin-labeled primers, and removing the biotin-labeled primers and hybridized molecular complexes thereof by using streptavidin magnetic beads to obtain enriched mRNA-cDNA;

the sequence of the r-cDNA probe is as follows: 5' -X _(20-30) Y _(10-15) Z _(20-30) -3', X, Y, Z is one of ATCG; x is X _(20-30) For hybridization with biotin-labeled primers; y is Y _(10-15) For separating X from Z to form a segment of non-hybridizing region; z is Z _(20-30) For hybridization with r-cDNA;

the sequence of the biotin-labeled primer is identical to that of the X of the r-cDNA probe _(20-30) Hybridization is performed.

2. The method for enriching mRNA-cDNA of bacteria according to claim 1, wherein: in the step (1), the sequence of the reverse transcription primer is as follows: 5' -X _(18-25) N ₆ -3'; wherein N is an ATCG random one, and X is confirmed as an ATCG one according to the primer sequences amplified by the subsequent library PCR.

3. The method for enriching mRNA-cDNA of bacteria according to claim 1, wherein: step (1) is directed to in situ labelled bacterial single cells or total bacterial RNA extracted in vitro.

4. The method for enriching mRNA-cDNA of bacteria according to claim 3, wherein: for in situ labeled bacterial single cells, the sequence of the reverse transcription primer is as follows: 5' -X _(10-15) N ₆ -3’；Wherein, N is one of ATCG and X is one of ATCG according to the primer sequence amplified by the subsequent library PCR; and carrying out phosphorylation modification at the most 5' end of the reverse transcription primer.

5. The method for enriching a bacterial mRNA-cDNA according to claim 3, wherein:

in the step (1), the method for obtaining total cDNA by reverse transcription of total RNA of bacteria is as follows:

1) A method for total RNA in vitro comprising the steps of: extracting total RNA of the escherichia coli, and carrying out reverse transcription on the total RNA by using a reverse transcription primer to synthesize total cDNA;

2) A method for labeling bacterial single cells in situ comprising the steps of: fixing, penetrating and digesting the bacterial cells with chromosome DNA, and then carrying out random reverse transcription in the bacterial cells by using reverse transcription primers to synthesize total cDNA.

6. The method for enriching mRNA-cDNA of bacteria according to claim 1, wherein: in the step (2), the sequence of the TSO primer is as follows: 5' -X _(25-30) rGrG+G-3', where X identifies it as one of the ATCG based on the subsequent library PCR amplified primer sequences.

7. Use of the enrichment method of bacterial mRNA-cDNA according to any of claims 1 to 6 for constructing a bacterial transcriptome sequencing library.

8. The use according to claim 7, characterized in that: including construction of single cell transcriptome sequencing libraries based on bacterial in situ markers and construction of conventional bacterial transcriptome sequencing libraries.

9. Use of the method for enrichment of bacterial mRNA-cDNA according to any of claims 1 to 6 for bacterial transcriptome sequencing.

10. The use according to claim 9, characterized in that: including single cell transcriptome sequencing based on bacterial in situ markers and conventional bacterial transcriptome sequencing.