RNA-seq of INS1 cell
mRNA was purified from approximately 200 ng of total RNA with oligo-dT beads and sheared by incubation at 94C in the presence of Mg (#KR1352, Roche). Following first-strand synthesis with random primers, second strand synthesis and A-tailing was performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3 dTMP overhangs were ligated to library insert fragments.  Library amplification amplifies fragments carrying the appropriate adapter sequences at both ends.  Strands marked with dUTP are not amplified. Indexed libraries were quantified by qRT-PCR using a commercially available kit (#KK4854, Roche) and insert size distribution determined by the Agilent 2100 Bioanalyzer. Samples with a yield of  0.5 ng/l and a size distribution of 150-300 bp were used for sequencing.