GSM3142908: wt_YA_short_cap_RNAseq_rep1; Caenorhabditis elegans; RNA-Seq
Nuclei were isolated and then chromatin associated RNA was isolated as in (Pandya-Jones and Black 2009), resuspending washed nuclei in Trizol for RNA extraction. Following purification, RNA was of 17–200nt was isoated using Zymo clean and concentrate columns. To profile transcription initiation ("short cap RNA-seq"), stranded libraries were prepared from 17–200nt RNA. Non-capped RNA was degraded by first converting uncapped RNAs into 5'-monophosphorylated RNAs using RNA polyphosphatase (Epibio), then treated with 5' Terminator nuclease (Epibio). The RNA was then treated with calf intestinal phosphatase to remove 5' phosphates from undegraded RNA, and decapped using Tobacco Acid Pyrophosphatase (Epicentre, protocol 1), Cap-Clip Acid Pyrophosphatase (CellScript, protocol 2) or Decapping Pyrophosphohydrolase, (Dpph tebu-bio protocol 3) and then converted into sequencing libraries using the Illumina TruSeq Small RNA Preparation Kit kit. Libraries were size selected to be 145–225 bp long on a 6% acrylamide gel, giving inserts of 20–100 bp long. Libraries were made from two biological replicates for each developmental stage. During the course of this work, the TAP enzyme stopped being available; the Cap-Clip and Dpph enzymes perform less well than TAP. One L3 replicate and one YA replicate were made using a slightly different protocol (protocol 4) from Chen et al 2013 (doi: 10.1101/gr.153668.112). RNA-seq of short (<100nt) capped chromatin associated RNA
GEO Accession
GSM3142908