GSM8111305: CLIP-seq.CM_NCBP2_rep1; Mus musculus; RIP-Seq
Neonatal CMs, ECs, or FBs (2.0×107) cells were UV-cross-linked at a dose of 300 mJ/cm2 at 254 nm, and harvested with a cell scraper. Cells were lysed in two volumes of radio immunoprecipitation assay lysis buffer (RIPA) containing 150 mM NaCl, 50 mM Tris-HCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 1 × protease inhibitor cocktail (Roche, 05892970001) for 30 minutes in a rotation wheel at 4°C. Lysate was cleared by centrifugation at 14,000 rpm for 15 min at 4°C. A volume of 20 μl lysate was saved as input. Ten micrograms of antibodies were washed with 1 ml ice-cold wash buffer for four times, and added to lysates followed by incubation for 14 hours on a rotation wheel at 4°C. Then, beads were added to the lysates, and incubation was continued for 4 hours on a rotation wheel at 4°C. Supernatant was removed and saved as flow-through, and beads were washed six times with 1 ml of ice-cold wash buffer. RNAs not bound by the protein was degraded using MNase (Thermo Fisher Scientific, N0181) for 10 min at 37°C. Afterwards, the products were washed, the 3' phosphate group was removed for 30 min at 37°C using FastAP (Life Technologies, EF0652), and RNA was ligated in a 3'App-DNA linker ligation assay for 60 min at 65°C using T4 RNA ligase 2 (NEB, M0351). The volume of 250 μl of elution solution was added to each sample and was rotated to elute. Then, the eluent, 50 μl of input protein, and 50 μl of flow-through protein were separated by SDS-PAGE (Thermo Fisher Scientific, NP0321BOX) and then transferred to a nitrocellulose membrane (Bio-Rad, 162-0115). The membrane was incubated in one volume of proteinase K solution including 100 mM Tris-Cl (pH 7.4), 50 mM NaCl, 20 mM EDTA and 5% NP-40 for 45 min at 55°C to release RNA. Then, the released RNA was extracted using an extraction buffer (Solarbio, SL2041-100mL), and 5'linker ligation was performed. Finally, modified RNA was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen, 18080093) and amplified using the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, F530S) for sequencing. The reverse transcribed cDNA was amplified using the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, F530S) and P5 and P7 primers including different indices combination for libraries construction.