GSM5729382: IL13: IL13 treated colonoid donor 5; Homo sapiens; RNA-Seq
Total RNA was isolated from human colonoids (released from Matrigel with Cell Recovery solution (Corning)) using RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. On-column DNase digestion was performed to remove any residual genomic DNA (Qiagen). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized 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, NEBNext Adaptors with hairpin loop structure were ligated. The library fragments were then purified with AMPure XP system (Beckman Coulter, Beverly, USA).