H2AK199Ub_MSQ43_Rep1
This ChIP-seq experiment used 2030 million MSQ43 cells fixed with 1% paraformaldehyde for 15 min at room temperature, quenched with glycine, and lysed in a buffer containing 10 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors, and 0.5% SDS. Chromatin was sonicated (Bandelin Sonopulse, 75% power, 8 cycles) and quantified via Qubit. For each ChIP, 2040 g chromatin was diluted to 0.10.2% SDS, pre-cleared with Protein A magnetic beads (NEB), and incubated overnight with antibody-bound beads (5 g antibody). Immunoprecipitated complexes were washed sequentially in high-salt (500 mM NaCl), LiCl-containing, and TE-based buffers. Cross-links were reversed, DNA eluted in SDS buffer at 65C for 14 hr, and purified using Zymo ChIP DNA Clean & Concentrator columns. Libraries were prepared with KAPA Hyper Prep Kit (Roche) and sequenced on a DNBSEQ platform. Key steps included RNase A and Proteinase K treatments for DNA cleanup.