RNA-seq Analysis of Liver and Muscle Tissues

Total RNA was isolated using the RNeasy minikit (Qiagen) (liver) or Qiazol followed by the RNeasy (muscle, fat). RNA quality was verified using a 2100 Bioanalyzer with RNA 6000Nano Reagents (Agilent Technologies).
Library preparation and rRNA depletion was performed using the Illumina TruSeq stranded/unstranded mRNA Library Prep Kit v2 chemistry in an automated system (Agilent Bravo liquid handling platform) starting with 1 μg total RNA for each sample. Libraries were sequenced on the Illumina HiSeq2500 or HiSeq4000. Sequencing quality was assessed with FastQC (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/). Reads were mapped to the mouse genome mm10 (Ensembl build 38.91) and reads per gene were counted using STAR version 2.4.2a⁴⁹ . Gene count normalization and differential expression analysis was performed using DESeq2^{50 (link)}. For gene annotation, biomaRt was used^{51 (link)}. Functional enrichment according to gene ontology was carried out using GOrilla^{52 (link)}. All scripts are deposited at github (https://github.com/FranziG/E47KO-liver). Sequencing data, normalized count data and DESeq2 output can be accessed via NCBI’s Gene Expression Omnibus using the accession number GSE111877.