GSM6245213: CUT&RUN E9.5 fibroblast, female, CTCF, rep1; Mus musculus; OTHER
Embryos were first resuspended in washing buffer (20mM HEPES-KOH pH 7.5, 150mM NaCl, 0.5mM spermidine and protease inhibitor) and then bound to the Concanavalin-coated magnetic beads (Polyscience, #86057) in binding buffer (20mM HEPES-KOH pH7.9, 10mM KCl, 1mM CaCl2 and 1mM MnCl2). The samples were incubated in antibody buffer (washing buffer plus 0.005%~0.01% digitonin and 2mM EDTA) with antibody diluted at ratio of 1:100 at 4°C on Thermomixer for 3 hrs at 300 rpm. Beads were then washed and incubated with protein A-MNase (pA-MNase) (to a final concentration of 400 to 700 ng/ml) at 4°C for 1-3 hrs on a thermomixer at 300 rpm. Targeted chromatin was digested for 30 min on ice after adding 2 ml of 100 mM CaCl2. The reaction was then terminated with 2× stop buffer (340mM NaCl, 20mM EDTA\, 4mM EGTA, 50μg/mL RNase A, 100μg/mL glycogen and 0.005%~0.01% digitonin). Samples were then incubated at 37°C for 20 min to release the targeted fragment. CUT&RUN libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, #E7645S)  following manufacturer's protocols. The libraries were purified and size-elected with AMPure XP beads (Beckman Coulter, #A63881).