GSM9512905: Spleen, CD23+ B cell, Xist tat KO, 0h naïve, H3K27me3, biol rep 1; Mus musculus; OTHER
Cells were collected at respective timepoint and immobilized on concanavalin A-coated beads in Wash Buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine, 1x Protease Inhibitor Cocktail). 3ug (H3K27me3) or 1.5ug  (H2AK119Ub) of antibody was incubated with immobilized cells at 4°C for 5 hours with rotation in Antibody Buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine, 1x PIC, 0.05% Digitonin, 2mM EDTA). Cells were washed then incubated with pA-MNase fusion protein at 4°C for 1 hour with rotation in Digitonin Wash Buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine, 1x PIC, 0.05% Digitonin). Digestion was performed in Digitonin Wash Buffer at 0°C for 30 minutes by adding CaCl2 to 2mM. The digestion was stopped by adding one volume of 2X STOP Buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.02% Digitonin, 50ug/mL RNase A, 50ug/mL Linear Acrylamide, 2pg/mL Yeast Spike-In DNA). Chromatin was released from cells by shaking beads at 300rpm at 37°C for 10 minutes. The supernatant was incubated at 70°C for 10 min with SDS to 0.1% and 50ug of Proteinase K. DNA was then isolated with phenol:chloroform:isoamyl alcohol followed by precipitation with ammonium acetate. Libraries were constructed with 5ng of CUT&RUN DNA using NEBNext Ultra II DNA Library Prep Kit for Illumina following the manufacturor's protocol