Method for constructing library on basis of RNA samples, and use thereof
Provided are a method for constructing a library based on an RNA sample and uses thereof. The method includes: step 1 of subjecting the RNA sample to a reverse transcription reaction to obtain DNA-RNA hybrid strands; step 2 of performing reaction of the DNA-RNA hybrid strands with an endoribonuclease, a first DNA polymerase, a second DNA polymerase, and dATPs to obtain a double-stranded DNA added with dA-tail, where the first DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity, and the second DNA polymerase has no 3′-5′ exonuclease activity; step 3 of ligating the double-stranded DNA added with dA-tail and a sequencing adaptor to obtain a ligated product; and step 4 of subjecting the ligated product to PCR amplification to obtain a sequencing library.
1 . A method for constructing a library based on an RNA sample, the method comprising:
step 1 of subjecting the RNA sample to a reverse transcription reaction to obtain DNA-RNA hybrid strands;
step 2 of performing reaction of the DNA-RNA hybrid strands with an endoribonuclease, a first DNA polymerase, a second DNA polymerase, and dATPs to obtain a double-stranded DNA added with dA-tailing, wherein the first DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity, and the second DNA polymerase has no 3′-5′ exonuclease activity;
step 3 of ligating the double-stranded DNA added with dA-tail and a sequencing adaptor to obtain a ligated product; and
step 4 of subjecting the ligated product to PCR amplification to obtain a sequencing library.
2 . The method according to claim 1 , wherein the endoribonuclease is RNase H.
3 . The method according to claim 1 , wherein the first DNA polymerase is DNA polymerase I; and the second DNA polymerase is selected from the group consisting of Taq DNA polymerase, Tth DNA polymerase, Bst DNA polymerase, Bst DNA polymerase of larger fragment, Klenow Fragment (exo-), and combinations thereof.
4 . The method according to claim 1 , wherein the reaction in step 2 comprises: reacting at 10° C. to 20° C. for at least 1 hour and then reacting at 70° C. to 80° C. for 10 to 30 minutes to obtain the double-stranded DNA added with A-tailing.
5 . The method according to claim 1 , wherein a buffer used in the reaction in step 2 comprises magnesium ions at a final concentration ranging from 5 mM to 40 mM, Tris-Cl having a pH value between 6.5 and 8.5, and sodium or potassium ions at a final concentration of less than 100 mM.
6 . The method according to claim 1 , wherein step 1 further comprises:
step 1-1 of mixing and treating the RNA sample with 5′ end-phosphorylated random primers at 80° C. to 95° C. for 5 minutes to 15 minutes to obtain a fragmented RNA product; and
step 1-2 of mixing the fragmented RNA product with dNTPs, actinomycin D, an RNase inhibitor, and a reverse transcriptase for the reverse transcription reaction to obtain a first strand cDNA product.
7 . The method according to claim 6 , wherein the reverse transcription reaction comprises 10 minutes to 15 minutes at 25° C. to 30° C., 10 minutes to 20 minutes at 45° C. to 55° C., and 10 minutes to 20 minutes at 70° C. to 75° C.
8 . The method according to claim 6 , wherein the random primers have a length of 6 to 8 random nucleotides.
9 . The method according to claim 1 , further comprising, prior to step 4: purifying the ligated product using magnetic beads.
10 . The method according to claim 1 , wherein the RNA sample is a total RNA sample, an oligo (dT)-enriched mRNA sample, or an rRNA-free RNA sample.
11 . A sequencing library, the sequencing library being constructed with the method according to claim 1 .
12 . A method for sequencing an RNA sample, comprising:
constructing a sequencing library based on an RNA sample with the method according to claim 1 ; and
sequencing the sequencing library to obtain sequencing information of the RNA sample.