GSM5416956: 103593-001-064; Mus musculus; RNA-Seq
Next generation sequencing (NGS) was performed was performed on liver and eWAT tissue samples. Briefly, total RNA was extracted from was extracted from eWAT using an Ambion RNAqueous total RNA isolation kit (Thermo Fisher Scientific, Waltman, USA) and from liver using the RNA-Bee total-RNA isolation kit. 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 NextSeq 500 V2.5 was performed according to manufacturer's protocols. A concentration of 1.6 pM of DNA was used yielding single end reads (1x 75bp). NextSeq control software 2.2.0 was used. Image analysis, base calling, and quality check was performed with the Illumina data analysis pipeline RTA3.4.4 and Bcl2fastq v2.20.