Method for making a cDNA library
This disclosure provides, among other things, a method for making a cDNA library. In some embodiments the method may comprise reverse transcribing mRNA to produce DNA:mRNA hybrids, treating the DNA:mRNA hybrids with RNAseH to produce mRNA fragments, and reverse transcribing the mRNA fragments.
1. A reagent system for producing cDNA comprising:
(a) a first oligod(T) primer;
(b) a reverse transcriptase that has been engineered to remove intrinsic RNAseH activity;
(c) an RNaseH; and
(d) a poly(A) polymerase
(e) a second oligod(T) primer, wherein the second oligod(T) primer comprises a 5′ tail.
2. The system of claim 1 , wherein the system further comprises:
(f) a 5′ adapter.
3. The system of claim 2 , further comprising:
(g) an RNA ligase.
4. The system of claim 1 , wherein the first oligod(T) primer is an anchored oligo(dT) primer.
5. The system of claim 1 , wherein the first oligod(T) primer does not have a 5′ tail.
6. The system of claim 5 , wherein the system further comprises:
(f) a 5′ adapter; and
(g) an RNA ligase.
7. The system of claim 6 , 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.
8. The system of claim 1 , wherein at least one of (b), (c) and (d) is in a composition that further comprises a non-naturally occurring buffering agent.
9. The system of claim 8 , wherein the non-naturally occurring buffering agent is Tris, HEPES, TAPS, MOPS, tricine or MES.