GSM8459788: MAF-811, Normoxia, 72 hours, IgG, rep1; Homo sapiens; OTHER
A total of 500,000 cells per reaction were harvested and captured using 10µL of pre-activated ConA beads (EpiCypher). Beads with attached cells were incubated at room temperature for 10 min to ensure complete adsorption. Subsequently, 50µL of antibody buffer (wash buffer supplemented with 0.1% digitonin and 2mM EDTA) was added:  H3K27me3 (MA5-11198, Thermo Fisher), and a negative control, IgG (EpiCypher, 13-0042K). Cells were incubated overnight on a nutator at 4°C and permeabilized using a 5% digitonin buffer. Next, 2.5µL/reaction pAG-MNase (Epicypher) was added and beads were gently resuspended by pipetting. The mixture was incubated for 10min at room temperature. Calcium Chloride, CaCl2 (100mM, 1µL/reaction) was added, followed by a 2-hour incubation at 4°C. After incubation, 34µL of Stop Master Mix was added, followed by a 10-minute incubation at 37°C. Tubes were quick-spun and placed on a magnet for slurry separation. Clear supernatants were transferred to 8-strip tubes for DNA purification. DNA was quantified using dsDNA High Sensitivity Assay kit (Thermo Fisher). Libraries were prepared with the NEBNext Ultra II DNA kit, according to the manufacturer's instructions (New England Biolabs). Final libraries were size-selected (200-600 bp) and analyzed on Bioanalyzer High Sensitivity DNA chips (Agilent) to confirm 200 to 400 bp fragment size range. Paired-end 150 bp sequencing of pooled barcoded libraries was carried out on the Illumina Novaseq 6000 platform by the Novogene (CA,USA) facility or functional genomics core (CU Anschutz)