GSM4396391: TENT4A_Replicate1; Homo sapiens; RIP-Seq
HepG2.2.15 cells on two 150 mm dishes were cross-linked with 0.1% paraformaldehyde (Pierce, 28906), harvested and lysed. Each 15 μg antibody (Normal mouse IgG, Santa Cruz, sc-2025; TENT4A, labmade; TENT4B, labmade) was conjugated to protein A and G sepharose beads (1:1 mix, total 20 μl, GE Healthcare, 17-5138-01 and 17-0618-01, respectively). Lysates were incubated with antibody-conjugated beads, and RNAs were purified from the eluates followed by DNaseI treatment and washing. 1 μg of input RNAs or RNAs from each sample were prepared for the libraries. rRNAs were depleted twice by Ribo-Zero kit (Illumina, TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat, 20020596). The rRNA-depleted RNAs were ligated to the 3′ adaptor and purified by 6% urea-PAGE gel (80–500 nt, corresponding to 50–470 nt RNAs which were fragmented by sonication) followed by 5′ phosphorylation, 5′ adaptor ligation, reverse-transcription and PCR amplification. The fCLIP-seq libraries were sequenced by paired-end run (151×151 cycles) on Illumina platform (MiSeq) with PhiX control library v3 (Illumina).