GSM5834951: Trizol-301KO-3-1; Mus musculus; RNA-Seq
Total RNA was harvested using Trizol reagent. Poly(A) RNAs were purified by using the Dynabeads mRNA purification kit and Poly(A) RNAs were fragmented at 94℃ for 5 min before reverse transcribed to cDNA with random primers. cDNA fragments were blunted and ligated to sequencing adaptors. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3'ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3µl USER Enzyme (NEB,USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index(X)Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.