GSM9015005: Mouse islets, Whole-body knockout control, Male, rep3; Mus musculus; RNA-Seq
Mouse islets were digested from pancreata with collagenase and purified by Histopaque gradiant centrifugation. The RNA extraction was performed on isolated islets via Qiagen silica gel spin column. 500 ng total RNA sample was enriched for mRNA by oligo (dT) selection followed by fragmentation using divalent cations under elevated temperature.  Double-stranded cDNA was synthesized using SuperScript II RT (Invitrogen, Carlsbad, California) and random primers for first strand cDNA synthesis followed by second strand synthesis using DNA Polymerase I and then RNAse H for mRNA removal.  Double-stranded cDNA was purified with SPRI beads (Beckman Coulter Genomics, CA), followed by incubation with Klenow DNA Polymerase and Adenine to add an 'A' base (Adenine) to the 3' end of the blunt phosphorylated DNA fragments.  DNA fragments were ligated to Illumina unique molecular index (UMI) adapters, which have a single 'T' base (Thymine) overhang at their 3'end.  Following ligation, cDNA was purified with SPRI beads, and PCR-amplified (13 cycles) with PhusionTM DNA Polymerase using Illumina's genomic DNA primer set and purified with SPRI beads.