title: GSM8477129: HCT116 cells, sgCtrl, rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated using the standard Trizol extraction method. In contrast to cells, tissues required repetitive grinding in a mortar with liquid nitrogen prior to RNA extraction, followed by lysis with trizol (Invitrogen). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). Firststrand cDNA wassynthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA).