title: GSM8585410: WT Ctrl 2; Mus musculus; RIP-Seq
library_construction_protocol: On day 7, iCLIP experiments were performed as described (doi: https://doi.org/10.1101/2021.08.27.457890). Briefly, iGCB cells were irradiated 1x with ultraviolet (UV) light for cross-linkage of protein-RNA (150 mJ/cm2; Stratalinker 2400). After washing cells in ice-cold PBS, cell pellets were snap-frozen in liquid nitrogen and stored at -80C.  Next, cells were lysed with 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630 (Sigma), 0.1% SDS, and 0.5% sodium deoxycholate lysis buffer, and sonicated (3x 15''). For each mCherry-ZFP36L1 sample, cell lysates from two age- and sex-matched mice were pooled together, which were selected to match total iGCB cell numbers to WT controls. Total protein concentration from extract supernatants was measured by Pierce BCA protein assay and 1.5mg protein were treated for 3 min at 37 °C with 4U Turbo DNAse (Ambion) and 0.4U RNase I (ThermoFisher Scientific). mCherryZFP36L1-RNA complexes were immunoprecipitated with 20μl RFP-Trap magnetic particles M-270 (Chromotek), which were previously washed 3x in lysis buffer, and rotating overnight at 4°C. Immunoprecipitates were separated by SDS-PAGE. RNA-protein complexes were transferred to a nitrocellulose membrane and isolated after cutting the areas from ~100 to ~140 kDa where mCherryZFP36L1 was expected to be cross-linked to RNAs. After protein digestion, RNA isolation and cDNA library preparation was carried out as described (doi: https://doi.org/10.1101/2021.08.27.457890) using the L3-IR-App adapter to visualize and retrotranscribe the RNA cross-linked to mCherryZFP36L1. Amplification of iCLIP cDNA libraries was done with 21 PCR cycles.
identifiers.namespace: GEO
identifiers.id: GSM8585410_r1