title: GSM3868459: IgG_PAR_iCLIP; Homo sapiens; RIP-Seq
library_construction_protocol: PAR-iCLIP was performed as described (Huppertz et al., 2014). Briefly, after incubating hESCs with 100 μM 4-thiouridine for 7 hr, cells were irradiated with 800 mJ/cm2 UV light (365 nm). After sonication in 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40 and 1 mM EDTA, lysates were incubated with 20 U of Turbo DNase (Invitrogen) and 100 U RNase I (Invitrogen) at 37 oC for 10 min, followed by immunoprecipitation with anti-Exosc4 antibody (Bethyl A303-775A) conjugated to Dynabeads Protein G (Invitrogen). Beads were washed with high salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM EDTA), incubated with T4 PNK (NEB) followed by 3' linker ligation. After labeling RNA with 32P, the protein-RNA complex was fractionated in a 10% NuPAGE Bis-Tris gel (Invitrogen) and transferred to nitrocellulose. After autoradiography, protein-RNA complexes were excised and RNAs eluted from the membrane. After reverse transcription using Superscript III (Invitrogen) and primers containing 9 nucleotide barcodes (4 nucleotides for sample discrimination and 5 nucleotides to identify PCR duplicates), cDNAs were gel-purified, circularized with Circligase II (Epicentre) and cut using BamHI and a BamH1site-containing complementary oligonucleotide. Linearized cDNA was amplified using Phusion High-Fidelity PCR Master Mix (NEB) and sequenced on Illumina NEXTSeq.