GSM5669520: PolyA_NC NHDF_TNF6h_h1; Homo sapiens; RNA-Seq
For PAR-CLIP, Cells were cross-linked with 5000 uJ/cm2 of UV light (312nm) and fractionated nuclei were lysed and ribonucleocomplex were immunoprecipitated with anti-FLAGAb. Fluorescently labeled adaptors were ligated to the RNA and separated by SDS-PAGE. cDNA were synthesized by reverse transcript and subjected to library prep. For PAR-CLIP, the cDNA was amplified by PCR and libraries were size selected by Pippinprep. For RNA seq, rRNA depletion was performed with the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (New England BioLabs) and sequencing library construction was performed with NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads (New England BioLabs) according to the manufacturer's instructions. Each sample was multiplexed with NEBNext® Multiplex Oligos for Illumina® (96 Index Primers) (New England BioLabs). For poly A plus RNA-Seq from human fibroblasts, RNA  was extracted using RNeasy Plus Mini Kit (Qiagen).  Purified RNA was poly  (A) enriched and sequenced by Novogene Co.Ltd (California, USA). PAR-CLIP-seq, RNA-seq