title: GSM8596705: DMSO, H3K27ac; Mus musculus; OTHER
library_construction_protocol: 100,000 murine leukemia cells, added with 1% of spike-in control cells (human 293 cells stably expressed with GFP-H2B), were harvested and centrifuged for 3 min at 600g at room temperature. Pellets were washed with Wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail). Concanavalin A coated magnetic beads were activated in Beads Activation buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2) and 10 µl of activated beads were added per sample and incubated at room temperature for 10 min. The unbound supernatant was removed, and bead-bound cells were resuspended in Digitonin 150 buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail; 0.01% Digitonin) containing 2 mM EDTA and a 1:50 dilution of the appropriate primary antibody. Primary antibody incubation was performed on a rotating platform overnight at 4 °C. The next day, samples were incubated with appropriate secondary antibody (diluted 1:50 in Digitonin 150 buffer) at room temperature for 30 min. Cells were washed using the magnet stand with Digitonin 150 buffer to remove unbound antibodies. A 1:200 dilution of pA-Tn5 adapter complex was prepared in Digitonin 300 buffer (0.01% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1× Protease inhibitor cocktail). After added to the sample, sample was incubated at RT for 1 h. Samples were washed in Digitonin 300 buffer to remove unbound pA-Tn5 protein. Next, cells were resuspended in Tagmentation buffer (10 mM MgCl2 in Digitonin 300 buffer) and incubated at 37 °C for 1 h. To stop tagmentation, 2.25 µL of 0.5 M EDTA, 2.75 µL of 10% SDS and 0.5 µL of 20 mg/mL Proteinase K was added to 50 µL of sample, which was incubated at 55 °C for 30 min, and then at 70 °C for 20 min to inactivate Proteinase K. DNA was purified with Ampure XP beads following manufacture's instruction and eluted in 10 mM Tris pH 8.0.  To amplify libraries, eluted DNA was mixed with a universal i5 and a uniquely barcoded i7 primer, with a different barcode for each sample and 2x PCR master mix. Post-PCR clean-up was performed using 0.9x volume of Ampure XP beads, and libraries were eluted in 30 µL 10 mM Tris pH 8.0. Sequencing was conducted using an Illumina Nextseq 550 High Output Kit v2.5.