| Construction Protocol | RNA-seq libraries were prepared using a strand-specific library preparation protocol based on an early version of the Illumina TruSeq Small RNA Sample Prep Kit. In brief, rRNA-depleted RNA was fragmented to an average size of ~200nt. Fragmented RNA was 3’-de-phosphorylated with Antartic phosphatase and 5’-phosphorylated with polynucleotide kinase; this treatment prepares RNA fragments for subsequent ligation of Illumina RNA adaptors to their 5’ and 3’ ends using a 3’-RNA ligase and a T4 RNA ligase, respectively. First-strand cDNA was produced using a primer specific for the Illumina 3’-adaptor. The library was amplified with 15 PCR cycles using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter). Library size distributions and concentrations were determined on a Bioanalyzer (Agilent). RNA-seq libraries were sequenced on an Illumina HiSeq 2000 instrument (apart from nmt1-rpb1 sequenced on Genome Analyzer II). |
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