CUT&RUN of mouse Tregs with anti-HA-OGG1
Gently resuspend and transfer 10 L Concanavalin A coated magnetic beads slurry into 100 L Binding buffer in a 1.5 mL tube and mix by pipetting. Place the tube on a magnet stand to clear. Withdraw the liquid completely, resuspend in 10 L Binding buffer. Cells were harvested, counted and centrifuged for 3 min at 600g at room temperature. Aliquots of cells 100000 cells, were washed twice in 1 mL Wash Buffer. Resuspend cells in 100 L Wash Buffer. 10 L of activated Concanavalin A coated magnetic beads were added and incubated at room temperature for 15 min. The unbound supernatant was removed and bead-bound cells were resuspended in 100 L Dig-wash Buffer and a 1:100 dilution of the appropriate primary antibody. Primary antibody incubation was performed on a rotating platform for overnight at 4 C. Cells were washed twice in 1 mL Dig-wash Buffer to remove unbound Primary antibody. Squirt 150 L of the PA/G-MNase in Dig-wash. Place on the tube rotator at 4 C for 1 hr. Wash twice in 1 mL Dig-wash Buffer to remove unbound PA/G-MNase protein. Place on the magnet stand to clear and pull off all of the liquid. Next, resuspended the cells in 100 L Dig-wash Buffer and insert tube in wet ice to chill down to 0 C. Remove each tube from ice, Squirt 2 L 100 mM CaCl2, with gently vortexing and immediately place on the tube rotator at 4 C for 30 min. Add 100 L 2X STOP Buffer and mix by gently vortexing. Incubate 30 min at 37 C to release CUT&RUN fragments from the insoluble nuclear chromatin. Place on the magnet stand to clear, transfer the supernatant containing digested chromatin to a fresh 1.5 mL microcentrifuge tube and proceed with Phenol Chloroform Extraction. Extracted DNA was processed for library generation using the QIAseq Ultralow Input Library Kit (QIAGEN) following the manufacturers protocol. Libraries were quantified with Bioptic Qsep100 Analyzer (Bioptic lnc.) and paired-end sequenced with read lengths of 150bp.