GSM9626195: iCLIP2, E14, Sam68, day0, rep3; Mus musculus; OTHER
Crosslinked cells were lysed in 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, and 0.5% sodium deoxycholate, supplemented with protease inhibitors, and sonicated using a Bioraptor Pico (Diagenode; 3 cycles, 30 sec ON/OFF). Subsequently, RNA was digested with RNase I (1:200 dilution, AM2295, Thermo Fisher Scientific) to generate RNA fragments of 50–200 nt SAM68 protein immunoprecipitation was performed using a SAM68-specific antibody (SAM68 Polyclonal Antibody; PA5-96507; Thermo Fisher Scientific) conjugated to Dynabeads Protein G beads (10004D, Thermo Fisher Scientific) for 1 h at 4°C. Protein–RNA complexes were visualized using 3'-end pCp-IR750 RNA labeling of 10% of the beads, while the remaining 90% underwent the first adapter ligation with the pre-adenylated L3-App oligo (Supplementary Table 2S). After SDS-PAGE and transfer to a nitrocellulose membrane, RNA–protein complexes were isolated from the membrane, treated with proteinase K (Merck), and RNA was extracted using the RNA Clean and Concentrator-5 kit (Zymo, R1016). cDNA was generated by reverse transcription using SuperScript III (18080085, Thermo Fisher Scientific); reverse transcription terminates at the crosslinked site, generating truncated cDNAs. A second adapter, containing a unique molecular identifier (UMI) and an experimental barcode, was ligated to the 3′ OH of the cDNAs. MyONE beads were used to purify the cDNA, and the library was subsequently subjected to two rounds of PCR amplification (6 and 15 cycles, respectively) and size-selected using ProNex chemistry (NG2001, Promega). Library quality was assessed using a TapeStation system with the High Sensitivity D1000 Kit.