RNA-Seq of cells from Islets of Pdx1CreERT/1; Yy1fl/fl (cKO) mice
Total RNA was isolated and analyzed on the Agilent Tape station for RNA integrity numbers (RINs) before library preparation. RNA-seq libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (NEB, Cat. No. E7775) according to the manufacturers protocol. mRNA was isolated using poly-T oligos conjugated to magnetic beads and then fragmented and reverse-transcribed to cDNA. Deoxyuridine triphosphates (dUTPs) were incorporated during second strand synthesis and thus were not amplified. cDNA underwent end-repair, ligation with indexed adapters, and PCR amplification. Nucleic acid was cleaned up after each step using AMPure XP beads (Beckman Coulter). Libraries were quantified, pooled, and sequenced at paired-end 150 bp on the Illumina HiSeq platform with a total of 80 million reads per library.