Rnaclean xp spri beads

RNA-seq libraries were constructed using the strand-specific dUTP method^{50 (link)}, with minor modifications. Input amounts of RNA varied between 3–5 μg with the proportional amount of ERCC spike in RNA (Ambion). For each batch experiment total amounts of input RNA were consistent across the samples. For poly(A)⁺ RNA, DNAse-treated RNA was enriched for poly(A)⁺ RNA using Dynabeads mRNA purification (Ambion). For strand-specific total RNA libraries, DNAse-treated RNA was depleted of rRNA using Ribo-Zero (Epicentre), then samples were cleaned up using the RiboMinus concentration module (Invitrogen) and fragmented at 90 °C for 3 min using NEB fragmentation buffer. First-strand synthesis was followed by cleanup with RNAClean XP SPRI beads (Agencourt). Second-strand synthesis incorporated dUTP, which was followed by sample clean up with MinElute PCR purification kit (Qiagen). Fragment ends were repaired, adenylated, and then ligated to True-seq barcoded adaptors and cleaned up with AMPure XP SPRI beads (Agencourt). The libraries were then amplified by PCR for 9–12 cycles and cleaned up with AMPure XP SPRI beads. Illumina sequencing (1× 50 bp read length) was performed on a HiSeq 2000.