Cut&tag Seq of BAF60a in wt Min6 cells
In brief, approximately 10E5 single cells were washed twice in 1 mL PBS. Cells were incubated with 10 L of activated concanavalin A coated magnetic beads (Bangs Laboratories, BP531) in 500 L Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, and protease inhibitor cocktail (Roche) for 10 min under room temperature. Cell-bound beads were collected and resuspended with 50 L Dig-Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail, 0.05% Digitonin) containing 2 mM EDTA, 0.1% BSA and a 1:50 dilution of the primary antibody (Rabbit anti-CTCF, Active Motif, 61311; or normal Rabbit IgG, Cell Signaling, #2729), and incubated at 4C overnight. A secondary antibody (Goat anti-Rabbit IgG, Sigma, SAB3700883) diluted at 1:50 in 100 L of Dig-Wash buffer was then administered into the beads and incubated for 60 min at room temperature following primary antibody removal with magnet stand (Vazyme, CM101). The preparation of the pG-Tn5 adapter complex was performed according to the manufacturers instruction with Hyperactive pG-Tn5 Transposase for CUT&Tag (Vazyme, S602). Standard tagmentation and amplification were performed as reported previously. Amplified DNA libraries were purified with VAHTS DNA Clean Beads (Vazyme, N411) and shipped for NGS sequencing by Annoroad Gene Technology (Beijing).