title: GSM5911445: InVivo_30.5dpc_2; Rattus norvegicus; RNA-Seq
library_construction_protocol: Total RNA was extracted using RNeasy Micro Kit according to the manufacturer's instructions and stored at -20°C until use. Genomic DNA was removed by incubation with Turbo Dnase. RNA-Seq libraries were generated from 400 ng of total RNA using TruSeq Stranded mRNA LT Sample Preparation Kit, according to the manufacturer's instructions. Briefly, following purification with poly(T)-oligo attached magnetic beads, mRNA was fragmented using divalent cations at 94°C for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis using DNA polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 seconds at 98°C; [10 seconds at 98°C, 30 seconds at 60°C, 30 seconds at 72°C] X 12 cycles; 5 minutes at 72°C) to create the cDNA library. The excess of PCR primers was further removed by purification using AMPure XP beads and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis. Libraries were then single-read sequenced with a length of 50 bp on an Illumina Hiseq4000 sequencer. Image analysis and base calling were carried out using RTA v.2.7.7 and bcl2fastq v.2.17.1.14.