Total RNA was isolated from purified cells using an AllPrep DNA/RNA Micro Kit (Qiagen, Hilden, Germany). RNA-seq libraries were prepared from 5 ng RNA per sample using the Ovation SoLo RNA-seq System (NuGEN, Redwood City, CA, USA). cDNA fragments of ∼350 bp were obtained by sonication (Bioruptor, Diagenode, Liège, Belgium), fluorescence-controlled PCR amplification, and size selection using AMPure XP Beads (Beckmann Coulter, Brea, CA, USA). Quality and mean fragment size of library samples were assessed using a Bioanalyzer (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA), and libraries were sequenced on Illumina sequencers in paired-end mode.
Sequencing data were analysed using the Galaxy platform.21 (link) RNA-seq reads were trimmed and mapped to the Mus musculus genome (NCBI37/mm9) using STAR.22 (link) After duplicate removal, transcript abundance was estimated as fragments per kilobase of transcript per million fragments, mapped using Cufflinks.23 (link)
Sequencing data were analysed using the Galaxy platform.21 (link) RNA-seq reads were trimmed and mapped to the Mus musculus genome (NCBI37/mm9) using STAR.22 (link) After duplicate removal, transcript abundance was estimated as fragments per kilobase of transcript per million fragments, mapped using Cufflinks.23 (link)
