title: GSM5215848: LUM0513 YAC128 mouse on FVN/B strain 7261183; Mus musculus; RNA-Seq
library_construction_protocol: Brains were isolated directly after sacrifice and brainstem, cerebellum, cortex and striatum were dissected and snapfrozen in liquid nitrogen. Approximately 30mg of tissue was used for RNA isolation, using Trizol and the PureLink RNA mini column kit (Thermo Fisher Scientific) according to manufacturers instruction. RNA was treated with DNAse to remove contamination of genomic DNA. Isolated RNA was shipped to DeCode Genetics (Reykjavik, Iceland) for RNA sequencing. Quality of RNA was assessed with the LabChip GX using the 96-well RNA kit (Perkin Elmer). Non strand-specific sample preparation was performed using the TruSeq Poly-A v2 kit (Illumina, San Diego, USA) following manufacturer's instructions. In brief, mRNA was captured using magnetic poly-T oligo-attached magnetic beads, RNA molecules were fragmented, and cDNA synthesis was performed using SuperScript II (Invitrogen, Carlsbad CA, USA) with random hexamer primers. Subsequently, 2nd strand cDNA synthesis was performed in conjunction with RNAse-H treatment. End repair was performed to generate blunt ends and 3′ adenylation was performed, followed by ligation of indexing adapters to the ds-cDNA. PCR was performed to amplify the fragments. Quality of sequencing libraries was determined through pool sequencing on a MiSeq instrument (Illumina) to assess insert size, sample diversity and optimize cluster densities. Pooled samples (4 samples/pool) were clustered on paired-end (PE) flowcells (1 pool per lane) using a cBot instrument (Illumina). The sequencing was performed using a HiSeq 2500 with v4 SBS sequencing kits (read lengths 2x125 cycles).