Illumina HiSeq 2000 paired end sequencing
Strand specific RNAseq libraries were generated using the method described by Parkhomchuk et al. with minor modifications. mRNA was isolated from 500 ng total RNA using oligo-dT Dynabeads (Life Technologies) and fragmented to 150-200 nt in first strand buffer for 3 minutes at 94 C. First strand cDNA was generated using random primers. Second strand was generated using dUTP instead of dTTP to tag the second strand. Subsequent steps to generate the sequencing libraries were performed with the NebNext kit for Illumina sequencing with the following modifications: after adapter ligation to the dsDNA fragments, libraries were treated with USER enzyme (NEB M5505L) in order to digest the second strand derived fragments. Amplified libraries were pooled and sequenced in one single lane. Paired-end (2x100 bp) sequencing was performed on the Illumina HiSeq2000.