GSM1464038: v65 polyA(+) icSHAPE DMSO Biological Replicate 2; Mus musculus; OTHER
Mock or icSHAPE modified cells were harvested using Trizol LS, total RNA purified using RNeasy columns and processed for icSHAPE library construction. Total or polyA selected RNA was first reacted with DIBO-Biotin (Life Technologies) to CLICK biotin to icSHAPE modified transcripts. RNA was then fragmented with RNA Fragmentation Reagent (Ambion) to sizes ranging between 25-150 nts. Fragmented RNA was 3’end repaired with T4 PNK (NEB) and 3’ adaptors ligated with T4 RNL2tr (NEB). cDNA synthesis was achieved with SuperScriptIII (Life Technologies) and icSHAPE modified transcripts were captured with MyOne C1 Streptavidin beads (Life Technologies). Enriched icSHAPE modified cDNAs were circularized and amplified using quantitative PCR before deep sequencing.