title: GSM7094267: ChIPRx-HCT116-THZ531-15min-pol2; Homo sapiens; OTHER
library_construction_protocol: Following HaloPROTAC3 treatment, cells were collected and lysed with Trizol reagent. Total RNA was extracted according to the manufacturer's instructions. ChIP-Rx experiments were performed as previously described (Li et al., 2021; Wang et al., 2021). Briefly, 1×107 human cells were spiked-in with 1-2×106 MEF cells and were fixed with 1% paraformaldehyde in PBS for 10 minutes, and quenched with 0.125 M glycine for 5 minutes. Fixed cells were used for nuclei isolation and were sheared using the Diagenode Bruptor Plus with the high-power mode for 25 cycles (sonication cycle: 30 seconds ON, 30 seconds OFF). The chromatin was immunoprecipitated with 5 μg individual antibodies and 15 μL of pre-blocked Protein A/G beads (Santa Cruz, Cat# sc-2003). After wash for three times with wash buffer (50 mM HEPES-KOH at pH 7.5, 300 mM LiCl, 1 mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate), the captured DNA was eluted and reverse-crosslinked with 200 μL of elution buffer (50 mM Tris-HCl at pH 8.0, 10 mM EDTA, 1.0% SDS, 200 μg/mL proteinase K) by incubating at 55°C overnight. The DNA was purified by phenol-chloroform extraction and ethanol precipitation.