polyC_run2
The sRNA sample was poly(C)-tailed using poly(A) polymerase (PAP) in the presence of CTP instead of ATP. The reaction was carried our for 2 h at 37C, then additional PAP was added and the sample was further incubated at 37C for another two hours. Then, 5'-PPP structures were removed using tobacco acid pyrophosphatase. Afterwards, a RNA adapter was ligated to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dG)-adapter primer and M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to about 20-30 ng/l using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis
SUB14703285
polyC_run2