RIP-seq of enriched endogenous Z-RNA fragments by Z22 antibody and recombinant ZBP1 Za protein

To identify the potential RNA ligand for Z-RNA formation in MC38 cells under oxidative stress conditions, we performed the RNase A protection assay followed by RNA sequencing. Briefly, MC38 cells were initially treated for 3 hours with Arsenite (150 uM), then rinsed with PBS, and RNA was extracted using TRIzol (Invitrogen) in accordance with the manufacturer's instructions. Then, after being subjected to DNase I (NEB) treatment at 37 degree Celsius for 20 minutes and inactivation with EDTA at 70 degree Celsius for 10 minutes, RNA was further extracted by phenol-chloroform. Purified RNA was then pre-incubated with Z-RNA (Z-22) antibody or control IgG (2 ug/ml) or recombinant WT or mutant ZBP1 Za protein (150 nM) at room temperature for 10 min in 20 mM HEPES pH 7.5, 50 mM NaCl, 2 mM MgCl2 and 2 mM DTT, followed by addition of RNase A (6 ng/ul, final concentration 2 ng/ul) and incubation for another 5 minutes at room temperature. The TRIzol reagent (Invitrogen) was used to inactivate the RNase A digestion, and the RNA extraction kit (TIANGEN) was used to purify the RNA according to the manufacturer's instructions. To remove small fragments resulting from RNase A digestion (less than 100 bp), the extracted RNA was further purified using the TIANquick mini purification kit (TIANGEN). The final RNA was used for the cDNA library preparation. For RIP-seq, the immunoprecipitated RNA was subjected to high-throughput sequencing using Illumina Nova 6000. The library was constructed through RNA random fragmentation, cDNA strand 1/strand 2 synthesis, end repair, A-tailing, ligation of sequencing adapters, size selection, and library PCR enrichment.