GSM4669640: fibro_target_hits-clip-seq_mir-9124_3; Homo sapiens; OTHER
Day 7 cells were harvested for HITS-CLIP following the published protocol (Moore et al., 2014) with modifications. Briefly, cross-linked cells came from 3 dishes were lysed in lysis buffer, digested with RQ1 DNase (Promega, Madison, WI) and diluted (1:1000) RNAseA (Thermo, Waltham, MA) respectively, and then the RISC complex containing bound miRNAs and mRNAs (﻿ternary complex) was immunoprecipitated overnight at 4 oC with mouse anti-Ago1/2/3 antibody 2A8 (Active Motif, Carlsbad, CA). The ternary complex was labelled 32P by ligating miRNA with 32P-labelled 3'-linker and then separated by NuPAGE™ gel (Thermo, Waltham, MA). The 130 kDa bands on the nitrocellulose membrane corresponding to ternary complex containing Ago:miRNA:mRNA were excised and used to extract RNA for sequencing library prep. RNA was prepared for sequencing using TruSeq Small RNA Library Preparation Kits (San Diego, CA).
GEO Accession
GSM4669640