Fragmenting RNA:DNA hybrids
This disclosure provides methods of fragmenting RNA:DNA hybrids. These methods can be used to improve preparation of RNA for next-generation sequencing.
1 . A method of fragmenting an RNA:DNA hybrid, the method comprising:
(i) cleaving the RNA of a RNA:DNA hybrid using a ribonuclease to produce single strand regions of the DNA; and
(ii) cleaving the single stranded regions of the DNA using a single-strand-specific nuclease to produce RNA:DNA hybrid fragments.
2 . The method of claim 1 , wherein the ribonuclease is a non-sequence-specific ribonuclease.
3 . The method of claim 2 , wherein the non-sequence-specific ribonuclease is an RNase.
4 . The method of claim 3 , wherein the RNase is RNaseH.
5 . The method of claim 1 , wherein the single-strand specific nuclease is nuclease P1 or nuclease S1.
6 . A method of preparing RNA for sequencing, the method comprising:
(i) converting the RNA into an RNA:cDNA hybrid;
(ii) fragmenting the RNA:cDNA hybrid into RNA:cDNA hybrid fragments using a ribonuclease and a single-strand-specific nuclease; and
(iii) converting the RNA:cDNA hybrid fragments into double stranded DNA.
7 . The method of claim 6 , wherein the RNA:cDNA hybrid fragments are 200-300 base pairs in length.
8 . The method of claim 6 , wherein the RNA:cDNA hybrid fragments are 100-1500 base pairs in length.
9 . The method of claim 6 , wherein fragmenting comprises:
(a) cleaving the RNA of the RNA:cDNA hybrid using the ribonuclease to produce single-strand regions of the cDNA; and
(b) cleaving the single-stranded regions of the cDNA using the single-strand-specific nuclease to produce RNA:cDNA hybrid fragments.
10 . The method of claim 6 , wherein (i) converting the RNA into an RNA:cDNA hybrid comprises converting using a reverse transcriptase and primers.
11 . The method of claim 10 , wherein the primers are poly-T primers, random primers, or target specific primers.
12 . The method of claim 6 , wherein converting the RNA:cDNA hybrid fragments into double stranded DNA comprises using a polymerase.
13 . The method of claim 12 , wherein the polymerase is an E. coli Pol I.
14 . The method of claim 6 , comprising combining the RNA:cDNA hybrid, the ribonuclease, the single-strand-specific nuclease, and a polymerase into a single reaction mixture.
15 . The method of claim 6 , wherein (ii) and (iii) occur contemporaneously.
16 . The method of claim 6 , further comprising A-tailing the double stranded DNA.
17 . The method of claim 6 , further comprising ligating sequencing adaptors onto the double stranded DNA.
18 . A method of preparing RNA for sequencing, the method comprising:
(i) obtaining a plurality of RNAs;
(ii) converting RNAs of the plurality of RNAs into RNA:cDNA hybrids using a reverse transcriptase and a plurality of primers that are complementary to the RNAs of the plurality of RNAs;
(iii) fragmenting the RNA:cDNA hybrids into RNA:cDNA hybrid fragments, the fragmenting comprising:
(a) cleaving the RNA of the RNA:cDNA hybrid using an RNaseH to produce corresponding single strand regions of the cDNA; and
(b) cleaving the corresponding single stranded regions of the cDNA using a nuclease P1 to produce RNA:cDNA hybrid fragments;
(iv) converting the RNA:cDNA hybrid fragments into double stranded DNAs (dsDNAs) using a polymerase;
(v) A-tailing the dsDNAs to produce A tailed dsDNAs; and
(vi) attaching sequencing adaptors onto the dsDNAs.