GSM8988860: Flag/HA-ZC3H7B-ParClip- replicate 1; Homo sapiens; RIP-Seq
HEK293 cells were cultured and incubated with 100 μM 4-thiouridine (4SU) for 16 hours to incorporate photoreactive nucleosides into nascent RNAs. Subsequently, cells were irradiated with 365 nm UV light to induce covalent crosslinking between RBPs and 4SU-labeled RNAs. Cell lysates were prepared, and the RBP of interest was immunoprecipitated using specific antibodies. The crosslinked RNA-protein complexes were treated with RNase T1 to fragment RNA, and the RNA fragments were radiolabeled at the 5' end. Complexes were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and the regions corresponding to the RBP-RNA complexes were excised. Crosslinked RNA fragments were ligated first to a 3′ adenylated adapter using Rnl2 (1–249) K227Q ligase. Ligation products were purified by 15% denaturing PAGE, visualized via phosphorimaging, excised, and ethanol-precipitated. A 5′ RNA adapter was then ligated using T4 RNA ligase I, followed by a second PAGE purification step. Reverse transcription was performed with SuperScript III using adapter-specific primers. Following alkaline hydrolysis of RNA, cDNA was amplified by PCR with primers corresponding to the 5′ and 3′ adapters. Optimal cycle numbers were determined empirically by pilot PCR. Final libraries (95–110 nt) were purified via agarose gel extraction and assessed on an Agilent TapeStation using High Sensitivity D1000 ScreenTape. Libraries were sequenced on an Illumina HiSeq 2500 platform with 50 bp single-end reads. Adapters used were compatible with Illumina small RNA kits.