title: GSM9663023: HepG2.2.15 cells, IgG(iCLIP); Homo sapiens; OTHER
library_construction_protocol: The samples were irradiated twice with 254 nm UV-C light on ice at 400mJ, and lysed on ice for 15 min with lysis buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 400 mM NaCl, 0.1% SDS, 0.5% Triton X-100 (Merck, cat. no. X100), 1×Protease Inhibitor (Thermo Fisher, cat. no. 87785), 200 U/mL RNasin Plus (Promega, cat. no. N2611) and 20 U/mL DNase I (NEB, cat. no. M0303S)). The lysate was further treated with 40 U RNase I (Thermo Fisher, cat. no. EN0601) and rotary incubated at 37°C for 5 min. The reaction was quenched by adding RNasin® Plus. The mixture was centrifuged at 4°C for 15 minutes at 15,000 g, and 2% of the supernatant was was taken for Input sample. The remain of supernatant was incubated with XRCC5,XRCC6 and PUS1 antibody for immunoprecipitation, rotating slowly at 4°C for 4 h. 10 μL protein A/G magnetic beads (Thermo Fisher, cat. no. 88802) was added and incubated with immune complexes for 2 h followed by successive washes. The 3' phosphate groups on the RNA were removed using FastAP enzyme (Thermo Fisher, cat. no. EF0654) to facilitate subsequent ligation steps. A 3' App-DNA linker is ligated to the RNA molecules using T4 RNA ligase (NEB, cat. no. M0204). The RNA-protein complexes were then separated by Nupage 4-12% Bis-Tris protein gels (Thermo Fisher, cat. no. WG1401BX10) electrophoresis and transferred to a nitrocellulose (NC) membrane. The NC membrane was cut to extract the RNA bound to the protein of interest. The protein-bound RNA was extracted from the NC membrane by incubation with proteinase K (NEB, cat. no. P8107) at 37°C for 20 min and subsequent TRIzol (Thermo Fisher, cat. no. 15596018CN) treatment. The purified RNA was subsequently concentrated, desalted, reverse transcribed. The resulted cDNA was recovered, ligated with a 3' UMI (Unique Molecular Identity) adapter, and amplified by PCR to generate sequencing libraries, which was sequenced on a DNBSEQ-T7 sequencer (MGI) using PE150 mode.eCLIP-seq experiment and high through-put sequencing and data analysis were conducted by Seqhealth Technology Co., LTD., Wuhan, China. The purified RNA was subsequently concentrated, desalted, reverse transcribed. The resulted cDNA was recovered, ligated with a 3' UMI (Unique Molecular Identity) adapter, and amplified by PCR to generate sequencing libraries, which was sequenced on a DNBSEQ-T7 sequencer (MGI) using PE150 mode.eCLIP-seq experiment and high through-put sequencing and data analysis were conducted by Seqhealth Technology Co., LTD., Wuhan, China.