GSM7156414: 104766-001-028R; Mus musculus; RNA-Seq
For next generation sequencing total RNA was isolated from snap-frozen liver tissue us-490 ing the RNA-Bee total-RNA isolation kit (Bio-Connect, Huissen, The Netherlands) and 491 was purified with NucleoSpin RNA Clean-up (Macherey-Nagel, Düren, Germany). RNA 492 concentrations were determined by Nanodrop 1000 (Isogen Life Science, De Meern, The 493 Netherlands) and RNA quality was evaluated with 2100 Bioanalyzer (Agilent Technolo-494 gies). Isolated RNA was used for generation of strand-specific mRNA-seq libraries for 495 next generation sequencing. Next Generation Sequencing was performed at Genome Scan (Leiden, the Netherlands). Strand-specific cDNA libraries were generated from total-RNA using oligo-dT magnetic beads, mRNA fragmentation, NEBNext 496 Ultra II Directional RNA Library Prep Kit (NEB #E7760S/L, New England Biolabs, Ips-497 wich, MA, USA) resulting in 300–500 bp amplified libraries/sample. Libraries were multiplexed, clustered, and sequenced on a NovaSeq6000 v1.5 system (Illumina, New England Biolabs, Inc. Ipswich, MA, USA), with a paired-end, 150 bp sequencing protocol, 20-40 million reads per sample and indexing. Clustering and DNA sequencing using the NovaSeq6000 v1.5 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.