Cross-linking immunoprecipitation (CLIP)
This protocol is extracted from research article:
Functional roles of antisense enhancer RNA for promoting prostate cancer progression
Theranostics, Jan 1, 2021; DOI: 10.7150/thno.51931

5 × 106 C4-2 cells treated with 100 µM 4-Thiouridine (4SU) for 8 h were washed with cold PBS one time and cells were irradiated once with 150 mJ/cm2 at 365 nm using a Startalinker 38. Cells were lysed in lysis buffer (50 mM Tris-HCL, pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, protease inhibitor cooktail and RNase inhibitors) with protease inhibitors (1 mL) and transferred to 1.5 mL microtubes. Lysate was partially digested by 1 U/µL RNaseT1/A for 15 min at 22°C. RNA was immunoprecipitated with DNMT1 or Flag antibodies and protein A/G beads for 16 h at 4°C. After washed for 6 times, RNA was phosphorylated by T4 PNK and ligated RNA between 3' and 5' ends by RNA T4 ligase. SDS-PAGE loading buffer was added and the mixture was incubated at 70°C for 10 min. After running the SDS-PAGE gel, the RNA-protein complexes were transferred from gel to a nitrocellulose membrane using a wet transfer apparatus (30 V for 1 h). The membrane with target protein was cut up, and the targeted membrane piece was incubated with Proteinase K for de-crosslink. After de-crosslink, RNA was reverse transcribed into cDNA and subjected to real-time qPCR analysis.

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