title: GSM5537302: OPC_repl1_6hr; Mus musculus; OTHER
library_construction_protocol: RNA was extracted with Trizol. A spike-in 5-EU labeled control was added to each time point prior to library preparation. Samples were DNAse treated, depleted of ribosomal RNAs and fragmented by alkaline treatment. After precipitation, biotinylation of the 5-EU labelled RNA was performed following the Click-iT nascent RNA capture kit protocol, using 0.5 mM biotin azide. After PAGE purification, pull-down was performed with C1 myOne streptavidin beads (Thermo Fisher). RNA was dephosphorylated on the beads, and ligated to a preadenylylated linker (Universal miRNA Cloning Linker) before performing reverse transcription on the beads. The cDNA was eluted from the beads, PAGE purified, circularized with CircLigase, and PCR was performed for 14 to 16 cycles. Libraries were sequenced on an Illumina HiSeq 2500 using single read 76 cycles runs.