GSM7733009: CTLA-4 and FMT rep 1; Mus musculus; RNA-Seq
2cm distal colon segments were taken from mice after confirmation of death, and placed into 1 ml of RNA later. RNA was extracted with Qiazol reagent (Invitrogen). Distilled solutions of RNA were purified using chloroform and isopropanol and eluted with ethanol before being resuspended in RNAse free water. RNA was cleaned up using the Qiagen RNeasy Microkit according to the manufacturer's guidelines. 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).