title: Transcriptome (Spike-in RNA-Seq) of mouse resting B cells (replicate 3)
design: To normalize transcriptome data we combined total RNA isolated from 10^6 resting or activated B cells with 1 μl of 1/10 dilution of Ambion’s ERCC RNA Spike-in Mix (92 mRNA standards). mRNA was then isolated and processed following Illumina’s RNA-seq protocol v2. Short reads generated by Illumina pipeline were aligned against mm9 using Tophat (ver. 1.0.12) (Trapnell et al., 2009) with default settings. Resulting alignments were analyzed using Cufflinks software (ver. 0.9.3) (Trapnell et al., 2010) provided with the annotation of Ensembl 60 genes (Hubbard et al., 2009) after removing germline antigen fragments, mitochondrial rRNA genes, miRNAs and small nuclear RNAs. No de novo transcript assembly was attempted since only single-end short read libraries were available.