GSM8919822: HepG2, CLIP-seq, input, siFBL, rep2; Homo sapiens; OTHER
For PAR-CLIP, two replicates of 150 million HepG2 cells were cultured with 200 μM 4SU for 14 hrs. Cells were crosslinked by 365 nm UV at 1500 J/m2 twice. For CLIP-seq, 3 replicates of 150 million HepG2 cells were crosslinked by 254 nm UV at 1500 J/m2 for three times. Samples were harvested and lysed by iCLIP lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1% NP-40 substitute, 0.1% SDS, 0.5% sodium deoxycholate, 100 × protein inhibitor cocktail and 100 × SUPERase•In™ RNase Inhibitor) at 4℃ for 15 mins with rotation. To release FUBP1 proteins associated with the chromatin, cell lysate was supplemented with 1% SDS, sonicated at 30% amplitude with 2s:4s cycles for 1 min on ice. The lysate was 10-fold diluted by iCLIP lysis buffer without SDS, centrifuged to save the supernatant, and treated with 0.2 U/μL RNase T1 (Thermo, EN0642) at 22℃ for 15 mins before quenched on ice for 5 mins. Protein G beads (Invitrogen, 10009D) were conjugated with 20 μg FUBP1 antibody (abcam, ab192867) by incubation at 4℃ for 1hr, then washed and mixed with RNase T1-treated lysate to rotate at 4℃ for 4 hrs. Beads were washed for three times with CLIP wash buffer (50mM Tris pH 7.5, 300 mM KCl, 0.05% NP-40 substitute, 1000 × protein inhibitor cocktail and 1000 × SUPERase•In™ RNase Inhibitor), digested by 10 U/μL RNase T1 at 22℃ for 8 mins, before they were washed by CLIP High salt buffer (50mM Tris pH 7.5, 500 mM KCl, 0.05% NP-40 substitute, 1000 × protein inhibitor cocktail and 1000 × SUPERase•In™ RNase Inhibitor) and PNK buffer without DTT for 3 times each and underwent end repair by T4 PNK (NEB, M0201L), then eluted by proteinase K digestion (Thermo Scientific, EO0491). For CLIP-seq, 2% input samples were harvested before incubation with antibody-conjugated protein G beads, treated with 10 U/μL RNase T1 at 22℃ for 8 mins, followed by proteinase K digestion and end repair in the next. Library was constructed by  NEBNext® Small RNA Library Prep Set (NEB, E7330S). PAR-CLIp was sequenced by Illumina NovaSeq 6000 with paired-end 50 bp. CLIP-seq was sequenced by Illumina NovaSeq X with single-end 100 bp.