Chromatin isolation by RNA purification (ChIRP)

38 20-mer anti-sense DNA probes with 3’BiotinTEG were designed at https://www.biosearchtech.com/support/tools/design-software/chirp-probe-designer and synthesized by GENEWIZ, Inc. (Suzhou, China). Chromatin isolation by RNA purification (ChIRP) was performed as previously described⁴³. Briefly, 6 × 10⁷ DIV9 primary neurons were treated with etoposide to induce DNA damage, and then were crosslinked with 1% glutaraldehyde for 10 min at room temperature and lysed in 1 ml lysis buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, 1% SDS, protease inhibitor, PMSF and RNA^OUT) followed by sonication for 20 min, 2 ml hybridization buffer (50 mM Tris-HCl, pH 7.0, 750 mM NaCl, 1% SDS, 1 mM EDTA, 15% formamide, protease inhibitor, PMSF and RNA^OUT) was added to the supernatant after centrifugation. Probes were added to the final concentration 50 nM, shaking overnight at 37°C for hybridization, 100 μl washed Dynabeads™ MyOne™ Streptavidin C1 (Thermo fisher scientific) beads were added to each tube for additional 1 h with shaking at 37 °C. Then beads were subjected to five washes with wash buffer (2× SSC, 0.5% SDS, protease inhibitor, PMSF and RNA^OUT). RNA was extracted from beads and input with Trizol followed by reverse transcription and qPCR. Proteins were eluted twice with elution buffer (2% trifluoroacetic acid (TFA) and 50% acetonitrile) at 50 °C with shaking and subjected to MS analysis.