GSM9668622: plx3397_1; Mus musculus; RNA-Seq
Total RNA was extracted using Trizol reagent. After total RNA was extracted, mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) with two rounds of purification. The cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA.