Size selection of RNA using poly(A) polymerase
This disclosure provides, among other things, a method for making a cDNA library. In some embodiments the method may comprise adding a polyA tail to the longer RNA fragments but not the shorter RNA fragments in a sample by incubating the population of RNA fragments with a polyA polymerase, wherein the reaction conditions used preferentially tail only the longer fragments but not the shorter fragments.
1. A method for processing an RNA sample, comprising,
(a) fragmenting a sample comprising RNA to produce a mixture of RNA fragments of different lengths, wherein the mixture of RNA fragments comprises longer RNA fragments and shorter RNA fragments,
(b) incubating the mixture of RNA fragments with a polyA polymerase under conditions wherein the polyA polymerase preferentially adds a polyA tail to the longer RNA fragments and not the shorter fragments, to produce tailed RNA;
(c) hybridizing the tailed RNA to an oligo(dT) oligonucleotide; and
(d) either (i) reverse transcribing the tailed RNA using the oligo(dT) oligonucleotide as a primer, to produce a cDNA library or (ii) enriching for the tailed RNA by washing away RNA molecules that are not hybridized to the oligo(dT) oligonucleotide.
2. The method of claim 1 , wherein the RNA sample comprises at least mRNA.
3. The method of claim 1 , wherein the longer fragments are at least 20 ribonucleotides in length and the shorter fragments up to 15 ribonucleotides in length.
4. The method of claim 1 , wherein the conditions of (b) comprise 0.6 mM to 1.2 mM of a divalent cation.
5. The method of claim 1 , wherein the conditions of (b) comprise 0.75 mM to 1.0 mM of a divalent cation.
6. The method of claim 4 , wherein the divalent cation is Mg 2+ .
7. The method of claim 1 , wherein the conditions of (b) comprise polyA polymerase at a concentration of less than 0.02 U/uL.
8. The method of claim 1 , wherein the conditions of (b) comprise polyA polymerase at a concentration in the range of 0.002 U/uL to 0.02 U/uL.
9. The method of claim 1 , wherein the sample of (a) comprises DNA:RNA hybrids, and the method comprises treating the sample with RNAseH.
10. The method of claim 1 , wherein (a) is done by heating the RNA in the presence of a divalent cation.
11. The method of claim 1 , wherein the RNA sample further comprises small RNAs, and:
the mixture of RNA fragments produced in (a) comprises: the small RNAs and the longer RNA fragments, and
the tailed RNA produced in (b) comprises tailed longer RNA fragments and tailed small RNAs.
12. The method of claim 11 , wherein the small RNAs include microRNA (miRNA) molecules, tiny non-coding RNA (tncRNA) molecules, small modulatory RNA (smRNA) molecules, Piwi-interacting RNA (pRNA) molecules, and snoRNA molecules.
13. The method of claim 1 , further comprising sequencing the cDNA library or the enriched tailed RNA.
14. The method of claim 13 , wherein the method comprises amplifying the cDNA library by PCR, prior to sequencing.
15. The method of claim 1 , wherein the method further comprises
adding an adaptor sequence to the 5′ end of the mixture of RNA fragments of (a) or the tailed RNA of (b).