GSM5224724: RB-CR75_S1 - IgG CUT&RUN replicate 2; Mus musculus; OTHER
CUT&RUN was carried out following the protocol given in Skene & Henikoff, 2017. Briefly, Concanavalin A magnetic beads were activated by washing twice in binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). 100000 FACS-sorted erythroblasts were washed twice in 1.5 ml of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and one Roche Complete Protease Inhibitor tablet) then resuspended in 200 ul wash buffer, mixed with 10 ul activated bead suspension and rotated for 5-10 min at RT. Tubes were then placed in a magnetic rack, the wash buffer was replaced with 200 ul of antibody buffer (wash buffer plus 0.02% digitonin, 2 mM EDTA and 1 ul Rabbit IgG antibody (Millipore; 12-370)) and the tubes rotated for 2 hours at 4 C. Tubes were then placed on a magnetic rack and the cells were washed once with digitonin buffer (wash buffer plus 0.02% digitonin) before being resuspended in 200 ul digitonin buffer. pA-MNase enzyme (a kind gift of the Henikoff lab) was added to each tube to a final concentration of 700 ng/ml and the tubes rotated for 1 hour at 4 C. Tubes were then placed on a magnetic rack and the cells were washed twice with digitonin buffer before being resuspended in 150 ul digitonin buffer. Digestion was initiated by the addition of 3 ul 100 mM CaCl2 to each sample and was allowed to proceed whilst tubes incubated on ice for 30 minutes before being quenched with 50 ul 4x stop buffer (680 mM NaCl, 40 mM EDTA, 8 mM EGTA, 0.04% digitonin, 0.1 mg/ml of RNase A, 0.1 mg/ml glycogen). Tubes were incubated for 10 min at 37 C, then centrifuged for 5 min at 4 C at 16,000 xg. The supernatant was moved to fresh tubes, mixed with 1 ul of 20% SDS and 1.5 ul of 20 mg/ml proteinase K, then incubated for 10 min at 70 C. DNA was purified by phenol-chloroform extraction and ethanol precipitation. Libraries were prepared using the Ultra-II DNA library prep kit (NEB) following the manufacturer's protocol with 13 PCR cycles.
GEO Accession
GSM5224724