GSM7989210: THP-1 cells, GSK-LSD-b-cat, CUT&RUN biol rep 2; Homo sapiens; OTHER
Briefly, Cell were washed in 1 mL of wash buffer (HEPES pH=7.5 20mM, Spermidine 0.5mM, NaCl 120mM) and centrifuged at 600g at room temperature for three times and the pellet were resuspended in 100 μL of wash buffer per reaction. Following this, 10 μL of activated Concavalin A beads per reaction were added, and the mixture were incubated at room temperature for 10 minutes with intermittent shaking. Captured cells were resuspended in 50 μL of antibody binding buffer (wash buffer with digitonin 0.05%, EDTA). Primary antibodies, including negative and positive controls, were added, and the tubes were nutated overnight at 4°C. On the following day, the samples were washed twice in dig wash buffer (Wash buffer with 0.05% digitonin), and a master mix of pAG-Mnase was prepared and added to each sample. After nutating at 4°C for 1 hour, the samples were washed to remove unbound pAG-Mnase. Tubes were cooled to 0°C for 5 minutes, then supplemented with CaCl2 to promote MNase digestion at 4°C for 2 hours followed by the addition of 2X STOP buffer (NaCl 340mM, EDTA 20mM, EGTA 4mM, digitonin 0.05%) and incubation at 37°C for 10-15 minutes. Following a brief spin and magnetic separation, the supernatant containing enriched target-bound chromatin was proceeded directly with DNA cleanup with Zymo DNA Clean&Concentrator-5 kit (Zymo research). DNA libraries were prepared by NEBNext Ultra II DNA library kit (E7645S) as per manufacturer's instructions. CUT&RUN DNA libraries were prepared by NEBNext Ultra II DNA library kit (E7645S) as per manufacturer's instructions, The supernatant containing enriched target-bound chromatin was proceeded directly with DNA cleanup with Zymo DNA Clean&Concentrator-5 kit (Zymo research). DNA libraries were prepared by NEBNext Ultra II DNA library kit (E7645S) as per manufacturer's instructions.