title: GSM9220662: mT3-2D, JQ1, BRD2; Mus musculus; ChIP-Seq
library_construction_protocol: For ChIP-seq, approximately 1 × 10⁷ cells were harvested per sample using TrypLE Express Enzyme (Gibco, 12605028) and resuspended in PBS to generate a single-cell suspension. Chromatin immunoprecipitation was carried out using the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, #9003) according to the manufacturer's protocol with minor modifications. Briefly, cells were fixed in 1% formaldehyde at room temperature for 10 minutes to crosslink protein-DNA interactions. Crosslinking was quenched with glycine (final concentration 125 mM) for 5 minutes at room temperature. Cells were then washed twice with ice-cold PBS and lysed in lysis buffer to isolate nuclei. Chromatin was enzymatically digested with 1 µl of micrococcal nuclease (MNase) at 37°C for 20 minutes to achieve partial digestion, followed by mechanical shearing using a Bioruptor (Diagenode) set to high power for 3 rounds of 10 cycles (30 seconds on, 30 seconds off). Shearing was performed in 1.5 ml TPX microtubes (Diagenode, C30010010) to generate DNA fragments ranging from 150 to 500 bp. DNA fragmentation was verified by 1.8-2% agarose gel electrophoresis. For immunoprecipitation, 10 µl of antibody was used per reaction: BRD2 (Cell Signaling Technology, 5848S) or BRD4 (Bethyl Laboratories, A301-985A100). Chromatin and protein G magnetic beads were incubated overnight at 4°C with antibodies under constant agitation using a tube rotator to promote optimal antigen-antibody interaction. Beads were then washed sequentially with low salt, high salt, and LiCl wash buffers, followed by TE buffer. Crosslinks were reversed by incubating with Proteinase K at 65°C for 2 hours. DNA was purified using spin columns included in the kit according to the manufacturer's instructions (Cell Signaling Technology, 14209S). Library preparation and sequencing for ChIP-seq was carried out by Novogene Co., LTD. Sample quality was assessed prior to library construction. DNA fragments were end-repaired, A-tailed, and ligated to Illumina adapters, followed by size selection, PCR amplification, and quantification.