GSM5530783: 50 μM BPS 24h_rep 4; Mus musculus; RNA-Seq
Total RNA was extracted using the Rneasy Mini kit (Qiagen). The concentration and purity of extracted RNA was assessed using NanoDrop 2000 spectrophotometer (Thermofisher). mRNA was purified from 1ug of total RNA using poly-T oligo-attached magnetic beads and fragmented randomly in a fragmentation buffer. First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-); second-strand cDNA was subsequently synthesized using DNA Polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of DNA fragments, NEBNext Adaptor was ligated to prepare for hybridization. The library fragments were purified with AMPure XP system. The final library was obtained by PCR amplification and purification of PCR products using AMPure XP beads.