RNA-Seq Library Preparation and Sequencing

3 μg of total RNA per sample were used for library preparation. Ribosomal RNA was depleted by using the GeneRead rRNA Depletion Kit (Qiagen). RNA fragmentation, cDNA synthesis and further RNA-Seq library preparation was done with the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). After enrichment and XP bead (Agencourt AMPure Kit; Beckman Coulter, Inc.) purification, quality control was done using Fragment Analyzer (Advanced Analytical). The bar-coded libraries were equimolarly pooled and subjected to 75 bp single-end sequencing on Illumina HiSeq 2000, resulting in an average of 33 million reads per sample. Sequencing raw data were deposited in GEO database under the GSE63948 accession number. The “Tuxedo Suite” of Bowtie, TopHat, Cufflinks and Cuffdiff [39 (link),40 (link),58 (link),59 (link)] was used for the alignment and expression analysis. We aligned the samples separately to the mm9 genome using the splice junction mapper Tophat (version 2.0.9), which used Bowtie 2 (version 2.1.0) for mapping. The Ensembl version 67 [41 (link)] was used as a support for the annotation during the alignment.