title: RNA-seq of NCM460 cell: intestinal epithelial cell
design: For library construction, ribosomal RNA was removed and poly (A)+ mRNA was purified with magnetic Oligo-dT beads (Invitrogen, USA). The purified mRNA was fragmented (200bp-305bp) using divalent cations under elevated temperature. These short fragments were copied into the first-strand cDNA using random hexamer primers and M-MuLV reverse transcriptase (Invitrogen, USA). Second-strand cDNA was synthesized by adding DNA Polymerase I and RNase H.