title: GSM4576259: RNA-seq_Wild type pancreas 3; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted from isolated islets with Nucleospin RNA plus XS. Complementary DNA was generated and linearly amplified from 3 ng total RNA using the Ovation RNA-seq V2 system (NuGEN technologies Inc., Leek, The Netherlands), according to the manufacturer's instructions. The amplified cDNA was then purified using Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France) in a 1.8:1 bead to sample ratio and fragmented by sonication using a Covaris E220 instrument (with duty cycle: 10%, maximum incident power: 175 watts and cycles/burst: 200 for 120 seconds). Complementary DNA were generated and linearly amplified from 6 ng of total RNA using the Ovation RNA-seq system V2 (NuGEN technologies Inc., Leek, Netherlands), according to manufacturer's instructions. Amplified cDNA was then purified using Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France) in a 1.8:1 ratio of beads to sample and fragmented by sonication using a Covaris E220 instrument (with duty factor: 10 percent, peak incident power: 175 watts and cycles/burst: 200 for 120 seconds). RNA-Seq libraries were generated from 100 ng of fragmented ds-cDNA using Ovation Ultralow Library System v2 (NuGEN technologies Inc., Leek, Netherlands) according to manufacturer's instructions with only 8 PCR cycles for library amplification. The final libraries were checked for quality and quantified using capillary electrophoresis.