title: GSM3704319: 103022-001-009_R1.fastq_MouseLiver_11_HFD; Mus musculus; RNA-Seq
library_construction_protocol: The mice were sacrificed at week 24 and the livers were isolated for histological evaluation of NASH parameters (as described above) [40] and lipid analysis. White adipose tissue depots were also collected and weighed. Gene expression analysis was carried out on liver tissue from eight mice in each group, selected as being representative of the group based on mean values of NASH parameters. All analyses were performed blindly. Total RNA from 8 animals per group was extracted from snap-frozen liver tissue using the Ambion RNAqueous total RNA isolation kit (Thermo Fisher Scientific, Inc.). The RNA concentration was measured using a NanoDrop 1000 spectrophotometer (Isogen Life Science, De Meern, Netherlands). RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Amstelveen, Netherlands). Samples were prepared in accordance with the manufacturer's protocol using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, Massachusetts, USA). mRNA was isolated from total RNA using the NEBNext Poly(A) mRNA magnetic isolation module (New England Biolabs). cDNA synthesis was performed and used for ligation with the sequencing adapters and polymerase chain reaction (PCR) amplification of the resulting product. The quality and yield of the PCR products were consistent with the expected size distribution (300–800 base pairs). The sequences were directly aligned with the mouse reference genome (GRCm38p4) using the Star2.5 algorithm with default settings. NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, Massachusetts, USA)