GSM8809005: part2, H_15; Mus musculus; RNA-Seq
Next generation sequencing (NGS) was performed in tot-RNA samples extracted from right liver lobe samples. RNA was isolated using the RNA-Bee total-RNA isolation kit (Bio-Connect, Huissen, the Netherlands) and evaluated for quality with the 2100 Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). NGS (NovaSeq6000) was performed at GenomeScan (Leiden, the Netherlands). mRNA was isolated from total RNA with oligo-dT magnetic beads. Next, cDNA was synthesized and subsequently ligated with sequencing adapters and amplified by PCR and was processed into tagged random sequence libraries (NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, NEB #E7760S/L , Biolabs) and sample quality was checked for proper size distribution (300-500 bp peak, Fragment Analyzer). The mixed (multiplex) sample libraries were sequenced on an Illumina NovaSeq6000 v1.5 sequencer (Illumina, San Diego) with a paired-read 150-cycle sequencing protocol at GenomeScan BV (Leiden, the Netherlands), resulting in ~20-50 million read counts per sample. Clustering and DNA sequencing using the NovaSeq6000 was performed according to manufacturer's protocols. A concentration of 1.1 nM of DNA was used yielding paired end reads (2x 150bp). 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.