GSM9219483: RNA-seq of HeLa cells arrested in metaphase by proTAME (replicate 1); Homo sapiens; RNA-Seq
Cell extract was sampled for RNA-seq and loaded over a density gradinet. Endogenous hnRNPC was immunoprecipitated for low- and highdensity fractions, and beads were treated with RNaseI, followed by dephosphorylation of the RNA fragment (CIP), 3' ligation using using T4 Rnl2(1–249)K227Q, and phosphorylation (T4 PNK). Flluorescent RNA-protein complex was separated by SDS-PAGE and excised. After proteinase K treatment, RNA was extracted and 5' end labelled with T4 RNA ligase. Following small RNA cDNA library preparation was performed. See Anastasakis et al., PMID:33503264. For fCLIP: Ligation products were reverse transcribed and amplified by PCR. For RNA-seq: RiboEX extraction; Ribo-Zero Gold depletion; TruSeq Stranded Total RNA Library Prep Gold.