title: GSM2107383: PFC_WT14_20_6; Rattus norvegicus; RNA-Seq
library_construction_protocol: RNA extraction was carried out using RNeasy lipid tissue kit (QIAGEN, Venlo, The Netherlands) 2.5 μg RNA was used for rRNA depletion using the Ribo‐Zero rRNA Removal Kit (Human/Mouse/Rat, Epicentre, Madison, Wisconsin, USA) according to the manufacturer's recommendations. RNA fragmentation reactions were performed using fragmentation buffer (5x; 200 mM Tris‐Ac, 500 mM potassium‐Ac, 150 mM magnesium‐Ac, pH 8.2) in a final concentration of 1x per reaction. Fragmentation reactions were incubated at 95°C for 4 min on a thermal cycler and placed on ice for 10 min. Fragmented rRNA-depleted RNA was purified using ethanol precipitation, First and second strand synthesis was performed as described by Kouwenhoven and colleagues (2015). samples were prepared for sequencing by end repair of 5 ng total DNA as measured by Qubit dsDNA HS (Invitrogen). NEXTflex adaptors (Bioo scientific, Austin, Texas, USA) were ligated to the DNA fragments, followed by post-ligation cleanup using Agencourt AMPure XP beads (Beckman Coulter, Woerden, The Netherlands), library amplification by PCR (10 cycles) and size selection (∼300 bp) using Agencourt AMPure XP beads (Beckman Coulter). Quality control of DNA libraries prepared for sequencing was performed by qPCR and by running the products on a Bioanalyzer (Bio-Rad, Veenendaal, The Netherlands). Cluster generation and sequencing (50 bp, single end reads) was performed with the Illumina HiSeq 2000 sequencer according to standard Illumina protocols. Samples were sequenced to a depth of approximately 29 million reads per sample. Reads were aligned to the rn4 rat genome assembly using the gsnap program