Strand-Specific RNA-Seq Library Construction

RNA-Seq libraries were constructed using the strand specific dUTP method [18] (link), with minor modifications. Briefly, 3 ug of DNAse treated RNA was depleted of rRNA using Ribozero (Epicentre). Two batches of rRNA-depleted samples were combined, cleaned by RiboMinus concentration module (Invitrogen) and fragmented at 90°C for 3 min (NEB fragmentation buffer). First strand synthesis was followed by cleanup with RNAClean XP SPRI beads (Agencourt). Second strand synthesis incorporated dUTP, followed by sample clean up with MinElute PCR purification Kit (Qiagen). Fragment ends were repaired, adenylated, then ligated to True-Seq barcoded adaptors and cleaned up with AMPure XP SPRI beads (Agencourt). The libraries were then amplified by PCR for 12 cycles and cleaned up with AMPure XP SPRI beads. Illumina sequencing (1×50 bp read length) was performed on a HiSeq 2000. 4SU libraries were prepared non-strand-specifically using standard Illumina RNA-Seq.