GSM8534096: MPP3, IgG, naive; Mus musculus; OTHER
tibias, femurs, and pelvic bones were isolated from mice and cell subpopulations (HSCs, MPP3s, GMPs, and Macrophages) were purified via FACS following cKit+ magnetic separation. Markers were for the following: HSCs (Lin-,cKit+, CD150+, CD48-), MPP3s (Lin-, cKit+, CD150-, CD48+, Flk2-, CD34+), GMPs (Lin-, ckit+, CD34+, CD16/32+), Macrophages (CD3-, B220-, NK1.1-, CD11b+, Ly6g-, Ly6c+). Cells were washed with wash buffer (50 mL  total, 20mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, and a Roche Complete protein inhibitor tablet), incubated with Concanavalin A-coated magnetic beads (Bang Laboratories L200731C) for 15 minutes at room temperature. Samples slurries were then magnetically separated, washed once more, and then incubated in primary antibody at respective dilutions (1:50 dilution for all) in wash buffer added with 0.05% Digtonin (Digitonin Wash Buffer) at 4C overnight. Samples were washed with Digitonin Wash buffer three times then incubated with pAG-MNase (142.5 Digitonin Wash Buffer + pAG-MNase, Epicypher 15-1016) for 1 hour at 4C. Slurries were then magnetically separated and washed three more times and resuspended in 150 uL Digtonin Wash buffer. 1 uL of 100mM CaCl2 was added to activate enzymatic cleaving, to which samples were stored on ice in 4C for 45 minutes. After time, one equal volume of 2X Stop buffer ( 340mM NaCl, 20 mM EDTA, 4mM EGTA, 0.05 Digtonin, 0.05 mg/mL glycogen. 5ug/mL RNase A, 2pg/mL heterologus spike-in DNA) was added to end the reaction. Samples were incubated for 30 minutes at 37C to release CUT&RUN fragments and then magnetically separated. Supernatant was collected for DNA fragments, and DNA was purified through phenol-chloroform extraction and ethanol precipitation. Samples were resuspended in molecular grade water for frozen until library preparation. DNA libraries were prepared for sequecning per standard Illumina protocols