GSM7068387: CD34 cells, healthy volunteer, replicate 1, H3K4me3CUT&RUN; Homo sapiens; OTHER
0.5 million K562 cells or 25,000 human CD34+ cells were harvested, washed, and bound to activated Concanavalin A-coated magnetic beads, then permeabilized with Wash buffer containing 0.05% Digitonin. The bead-cell slurry was incubated with 0.5 μl antibody in a antibody buffer at 4°C overnight on a nutator. After 3 washes in 1 ml Dig-wash, beads were resuspended in digitonin wash, mixed with pAG/MNase and incubated for 1 hr at room temperature. After two washes in Dig-wash, beads were resuspended in digitonin wash. Tubes were chilled to 0°C and 1 µl 100 mM CaCl2 was added while gently vortexing. Tubes were nutated at 4°C for 2 hours followed by addition of EGTA-STOP buffer. Beads were incubated at 37°C for 30 min, replaced on a magnet stand and the liquid was removed to a fresh tube and DNA was extracted. DNA quality and size distribution were checked on Fragment Analyzer. 10 ng of DNA was used for library preparation according to NEBNext® Ultra II DNA Library Prep Kit