GSM6249191: Infect WT HSC using antibody against mouse IgG [Cut&Run]; Mus musculus; OTHER
Leg and hip bones were isolated and HSCs were purified via FACS sorting following mechanical isolation and c-kit+ magnetic cell enrichment. HSCs (LK CD150+ CD48-) were washed with Wash Buffer (50mL total with 20 mM HEPES pH 7.5, 150mM NaCl, 0.5 mM Spermidine, and one Roche Complete protein inhibitor tablet) and bound to Concanavalin A-coated magnetic beads (Bang Laboratories L200731C) for 15 minutes room temperature. Sample slurries were magnetically separated, washed, and incubated with primary antibody diluted in wash buffer containing 0.05% digitonin (Dig Wash) overnight at 4 °C. Slurries were then magnetically separated and washed with Dig Wash three times and then incubated with protein A-MNase (pA-MN, Epicypher 15-1016) for 1 hour at 4 °C. Slurries were magnetically separated and washed, resuspended in 200uL Dig Wash, and placed on an iced heating block. 2uL of 2mM CaCl2 was added to catalyze the pA-MN reaction and digestion. After 45 minutes, one equal volume of Stop Buffer (340mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 0.05 mg/mL glycogen, 5 ug/mL RNase A, 2 pg/mL heterologous spike-in DNA) was added to end the reaction, and sample slurries were then incubated for 30 minutes at 37 °C to release fragments and then magnetically separated. The supernatant was collected, and DNA was purified via phenol-chloroform extraction and ethanol precipitation. Samples were resuspended in molecular-grade water after ethanol precipitation. DNA libraries were prepared for sequencing using standard Illumina protocols