RNA-seq of mus musculus: Sk. Muscle
Total RNA was purified from all samples using MagMAX-96 for Microarrays Total RNA Isolation Kit (Ambion) according to manufacturers specifications. Genomic DNA was removed using Qiagen DNase kit. mRNA was purified from total RNA using Dynabeads mRNA Purification Kit (Invitrogen). Strand-specific RNA-seq libraries were prepared using KAPA mRNA-Seq Library Preparation Kit (Kapa Biosystems). Twelve-cycle PCR was performed to amplify libraries. Sequencing was performed on Illumina HiSeq2500 (Illumina) by multiplexed single-read run with 33 cycles. Raw sequence data (BCL files) were converted to FASTQ format via Illumina bcl2fastq v2.17. Reads were decoded based on their barcodes and read quality was evaluated with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped to the mouse genome (NCBI GRCm38) using ArrayStudio software (OmicSoft, Cary, NC) allowing two mismatches. Reads mapped to the exons of a gene were summed at the gene level. Genes were flagged as detectable with minimum 10 reads. Differentially expressed genes were identified by DESeq2 package and significantly perturbed genes were defined with fold changes no less than 1.5 in either up or down direction and with p-values of at least 0.01.