GSM6045304: FIP2, Distal colon, rep3; Mus musculus; RNA-Seq
Epithelial cells were mechanically isolated from colon using micro forceps in ice-cold PBS containing 30 mM EDTA and immersed in TRIzol Reagent (Invitrogen 15596018). Libraries were prepared by Novogene Corporation Inc. (Sacramento, CA). mRNA is purified from total RNA using poly-T oligo-attached magnetic beads. The mRNA is first fragmented  randomly by addition of fragmentation buffer. Then first strand cDNA is synthesized using random hexamer primer and M-MuLV Reverse  Transcriptase (RNase H-). Second strand cDNA synthesis is subsequently performed using DNA Polymerase I and  RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified doublestranded  cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA  fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization(1). In order to select  cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system  (Beckman Coulter, Beverly, USA). Finally, the final library is gotten by PCR amplification and purification of PCR  products by AMPure XP beads.