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 reagent system for making cDNA from fragmented RNA comprising:
(a) a first oligod(T) primer;
(b) a reverse transcriptase;
(c) a thermostable RNaseH;
(d) a poly(A) polymerase;
(e) a 5′ adapter;
(f) an RNA ligase; and
(g) a second oligod(T) primer, wherein the second oligod(T) primer comprises a 5′ tail.
2. The system of claim 1 , wherein the RNaseH is active at a temperature in the range of 60° C. to 80° C.
3. The system of claim 1 , wherein the first oligod(T) primer is an anchored oligo(dT) primer.
4. The system of claim 1 , wherein the first oligod(T) primer does not have a 5′ tail.
5. The system of claim 1 , wherein the system further comprises:
(h) a pair of PCR primers, wherein the pair of PCR primers comprises a first primer that hybridizes to the 5′ tail of the second oligod(T) primer and a second primer that hybridizes to the complement of the 5′ adapter.