GSM9032977: MV411-shLuc-1_ChIP_input_DMSO_72h_REP1_230927_GTCCGC_S1; Homo sapiens; ChIP-Seq
First, cells were crosslinked with disuccinimidyl glutarate (DSG) by resuspending 20 million cells in 2 mL room-temperature PBS and adding 0.5 M DSG to a final concentration of 2 mM. The reaction was rocked at room temperature for 30 minutes, then quenched by pelleting the cells via centrifugation and washing twice with ice-cold PBS. Next, cells were resuspended in PBS and crosslinked with 2% formaldehyde for 8 minutes at room temperature with rocking. Formaldehyde was quenched by sequentially adding Tris-HCl (pH 8.0) to a final concentration of 100 mM, followed by glycine to a final concentration of 125 mM. Crosslinked cells were washed twice with PBS. Cells were then lysed by centrifuging for 15000 rpm for 10 minutes in SDS buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM EDTA, and 1% SDS, supplemented with protease inhibitors. The opaque pellet was resuspended in cold IP buffer (1/3 Triton dilution buffer and 2/3 SDS buffer). Chromatin was sonicated using a Covaris sonicator, with optimized settings to shear DNA to fragments between 150–1000 bp. The sonicated chromatin was clarified by centrifugation at 15,000 x g for 10 minutes at room temperature. A small amount of the resulting input after sonication was de-crosslinked and quantified using Qubit, and immunoprecipitations (IPs) were normalized to equal amounts of DNA. Protein A Dynabeads were prepared by washing in ChIP dilution buffer and blocking with BSA. Antibodies specific to, manufacturer listed above, Menin (5 µg per IP), KMT2A (10 µg per IP) IKAROS (Cell signaling Technology, 14859, 5ug per IP), MEIS1 (5ug per IP) and UBR5 (10 µg per IP) were conjugated to the beads by incubation at 4°C overnight. For Menin, KMT2A, IKAROS, and MEIS1, 20-30 µg of sheared chromatin per sample was loaded into each IP. 50 ug of sheared chromatin was loaded for each UBR5 IP. The sheared chromatin was diluted 2X with ChIP dilution buffer, then incubated with the antibody-conjugated beads at 4°C overnight on a rotating platform. Beads were washed 6 times sequentially with 3 different buffers, 2X washes with Mixed Micelle Wash buffer (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 5 mM EDTA, 5.2% Sucrose, 1.25% Triton X-100, 0.2% Sodium Dodecyl Sulfate), 2X washes with Buffer 500 (0.1% w/v deoxycholic acid, 1 mM EDTA, 50 mM Tris-HCl ph 8.0, 1% Triton X-100), and 2X washes with LiCl Detergent Wash Buffer (0.5% w/v deoxycholic acid, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 10 mM Tris-HCl ph=8) followed by two washes with 1× Tris-EDTA buffer (10 mM Tris pH 8.0, 1 mM EDTA). Chromatin was then eluted in elution buffer (50 mM Tris-HCl at pH 8.0, 10 mM EDTA, and 1% SDS). De-crosslinking of chromatin was performed overnight at 65°C in a buffer containing 100 mM NaHCO₃, 100 mM NaCl, and 1% SDS. DNA was purified using AMPureXP beads, following the manufacturer's recommendations, with a 1.2X bead-to-sample ratio. DNA concentration was quantified using a Qubit fluorometer, and fragment size was verified using a Tapestation system. All buffers, including SDS buffer, Triton dilution buffer, and mixed micelle wash buffer, were prepared according to standard formulations detailed in the supplementary section. ChIP and input DNA were processed for library preparation using the ThruPlex DNA-Seq kit (Takara Bio), employing single-index barcodes and 12–14 PCR cycles for amplification. Libraries were sequenced as paired-end reads (37 bp) on the NextSeq500 system (Illumina).