GSM5202020: LUM0381 FVN/B wildtype mouse 7263460; Mus musculus; RNA-Seq
For RNA sequencing, 200 µl of blood was obtained by tail cut at 40 and 75 weeks of age. Blood samples for RNA sequencing were collected in RNAprotect animal blood tubes (Qiagen) following manufacturer's instructions, stored overnight at 4°C and subsequently frozen at -80°C until RNA isolation. After thawing, filled blood tubes were incubated for 4 hours at 25°C to ensure proper cell lysis. Isolation of RNA was subsequently performed using the RNeasy protect animal blood kit (Qiagen, Hilden, Germany) according to manufacturer's instructions for total RNA isolation including DNAse treatment, resulting in isolation of RNA molecules longer than approximately 200 nucleotides. Reduction of alpha and beta globin mRNA was performed on RNA samples using the GLOBINclear magnetic bead kit for mouse/rat (Qiagen) following manufacturer's instructions. 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).