Cells at 10e6 cells/mL were fixed in 1% formaldehyde for 10 minutes at room temperature. Fixation was quenched with .125M glycine for 5 minutes. Cells were then spun down at 1200 rpm for 4 minutes, and the pellet washed 3 times in cold PBS. The cells were then re-suspended in swelling buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40) containing protease and phosphatase inhibitors, and incubated for 10 minutes in ice. At this point, the cell suspension was transferred to a glass Dounce homogenizer with a ‘tight’ pestle and the cell membrane broken with 25 strokes of the pestle. Nuclei were spun down at 3000xg for 5 minutes at 4 degrees. Nuclei were re-suspended in sonication buffer (50 nM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS) at a concentration of ~10e6 cell nuclei per mL sonication buffer. Nuclei were sonicated with a Branson probe sonicator for 3 minutes total, 10 seconds on and 45 seconds off, at 30 mA amplitute. Following sonication, the lysate was spun down at 16,000xg for 15 minutes to remove the insoluble material, and this step repeated and the supernatant retained (the ‘chromatin’) for ChIP. 5% of the chromatin lysate was frozen to serve as input material. The rest was used in immunoprecipitation with the following antibodies. 1 mL (about 10e6 cells of starting material) of chromatin was used for each IP. Antibodies and amount used were as follows: PU.1 (sc-352x, 6 ul), p300 (sc-585x, 10 ul), H3K27me3 (07-449, 3 ul), H3K4me1 (Diagenode C15410037, 5 ug) H3K4me2 (04-790, 2 ul), H3K4me3 (05-1339, 5ul). 50 ul of protein-G Dynabeads were added with the chromatin and antibody mixture, and tubes were places on a 360 rotor at 4 degrees overnight. The following morning, the IP was washed with the following buffers (all at 4 degrees, except for TE which is at room temperature): 1X in sonication buffer, 1X in wash A (50 mM HEPES pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), 1X in wash B (20mM Tris pH 8.0, 1mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate), 2X in TE (10mM Tris pH 8.0, 1mM EDTA). Chromatin was then eluted from the washed beads by incubating with 250ul elution buffer (1% SDS, 0.1M NaHCO3) for 15 minutes at room temperature. This step was repeated, and the eluted fractions combined for a total of 500 ul. To this, 20 ul 5M NaCl was added, and the samples incubated at 65 degrees for 4 hours to reverse the crosslinks. Afterwards, to remove the protein in the sample, 10 ul of 0.5M EDTA, 20 ul of 1M Tris pH 6.5, and 2 ul of 10mg/ml Proteinase K was added and incubated for 1 hr at 45 degrees. DNA was recovered by Phenol/chloroform extraction and quantified on a Qubit® 3.0 Fluorometer. 10 ng of ChIP DNA was used for each library. The NEBNext DNA library Prep Reagent set for Illumina (E6000L) was used to generate the libraries, along with the standard Illumina barcoding primers, following the manufacturers instructions.