GSM5416814: s103566-001-066; Mus musculus; RNA-Seq
RNA extraction was performed as described previously in detail [29]. Total RNA was extracted from individual liver (lobus dexter lateralis) samples (n=10-11/group), perigonadal WAT samples (n=8-10/group) and quadriceps (n=7-10/group) samples using glass beads and RNA-Bee (Campro Scientific, Veenendaal, The Netherlands). RNA integrity was examined using the RNA 6000 Nano Lab-on-a-Chip kit and a bioanalyzer 2100 (Agilent Technologies, Amstelveen, The Netherlands). To assess the quality of the samples, the concentration of the sample was determined using the Fragment Analyzer. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used to process the sample(s). The sample preparation was performed according to the protocol "NEBNext Ultra II Directional RNA Library Prep Kit for Illumina" (NEB #E7760S/L). Briefly, mRNA was isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplicification of the resulting product. The quality and yield after sample preparation was measured with the Fragment Analyzer (see appendices). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Clustering and DNA sequencing using the Illumina Nextseq 500 V2 or NovaSeq6000 v1.5 was performed according to manufacturer's protocols. A concentration of 1.6pM (NextSeq) or 1.1 nM (NovaSeq) of amplicon DNA was used yielding 15-25 million sequencing clusters per sample as: 2x 150 bp Paired-End reads (PE) for Muscle or 1x 75bp Single-End reads for Liver and Perigonadal WAT. NovaSeq control software NCS v1.7 was used. Image analysis, base calling, and quality check was performed with the Illumina data analysis pipeline RTA3.4.4 and Bcl2fastq v2.20.