RIP-qPCR assay was performed as previously described.41 (link) Briefly, neutrophils (~3 × 107 cells for each sample) were harvested and lysed in IP lysis buffer (Thermo Scientific) and then incubated with 10 μg anti-ALKBH5 antibody (Sigma) or 10 μg control anti-IgG antibody (Millipore) at 4 °C overnight. Then, the cell lysates were mixed with protein A/G beads (Thermo Scientific) at 4 °C for 2 h. The beads were washed 6 times using IP lysis buffer and then resuspended in proteinase K to incubate at 56 °C for 1 h. The immunoprecipitated and input RNAs were isolated using the TRIzol reagent for further RT-qPCR analysis.
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