CUTNRUN of mus musculus: HSPCs (LSK) bone marrow
CUT&RUN was performed as previously described3,4. 1x10^6 HPC5 cells were harvested from culture and 50-100,00 LSK cells were isolated from mouse bone marrow as previously described. After three rinses each with 1-ml wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and protease inhibitors) followed by 3 min 600 g spin at RT, cells in 1 ml wash buffer were captured by Concanavalin A-coated magnetic beads (10 l per million cells; Bangs laboratories, Inc.) at RT for 10 min with constant rotation. Upon three more washes, captured cells were permeabilized in 100 to 300 l of the same wash buffer plus 0.05% digitonin and 2 mM EDTA with simultaneous antibody binding (purified anti-ETV6 antibody and corresponding amounts of IgG control) at 4C overnight. Permeabilized cells/nuclei retained on the beads were washed 3 times with the same digitonin-wash buffer before incubation with 150 l of Protein A-fused Micrococcal Nuclease (pA-MNase; 700 ng/ml) at 4C for 1 h. After washing away unbound pA-MNase, antibody-associated protein-DNA complex was released from chromatin by adding CaCl2 to digitonin-wash buffer for a final 2 mM CaCl2 concentration and incubated at 0C (i.e., ice-water bath) for 30 min. Equal volume of 2x stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% digitonin, 100 g/ml RNase A, 50 g/ml glycogen) containing spike-in yeast DNA (20 pg/ml) was then added to the reaction with an additional 30-min incubation at 37C. The released DNA was isolated by proteinase K treatment and phenol/chloroform extraction and ethanol precipitation4. NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) was used to prepare CUT&RUN libraries. Barcoded libraries were sequenced using NextSeq 500 High Output.