title: GSM4308135: SMARCA4_30min_cutnrun; Homo sapiens; OTHER
library_construction_protocol: MNAse digest 200.000 single cells were harvested from human small intestinal organoids. After two washes, cells were bound to 10 µl of Concanavalin A-coated beads (Bangs Laboratories, cat. no. BP531), diluted in 1ml of binding buffer and rotated 10 min at RT. Cells were cleared on a magnetic rack and beads were resuspended in 50 µl antibody solution. The antibody anti-SMAD3 (1:50, abcam28379; RRID: AB_2192903) was used, and incubated on a rotator overnight at 4˚C. Beads were cleared on a magnetic rack and washed in digitonin buffer before resuspension in 50 µl of digitonin buffer with 700 ng/µl pA-MNase, in order to bind the fusion protein to the antibody. After 1h rotating at 4˚C, beads were cleared on a magnetic rack and washed twice. To activate MNase activity, beads were resuspended in 150 µl digitonin buffer and chilled down to 0˚C before 3 µl of 100 mM CaCl2 were added for 30 min at 0˚C. Reaction was stopped by adding 150 µl of 2X Stop buffer and tubes were incubated for 10 min at 37˚C to release chromatin. After centrifugation for 5 min at 4˚C at 16 000g, tubes were placed on a magnetic rack in order to remove beads. The supernatant was collected and purified using the NucleoSpin PCR clean up kit. DNA was stored at -20˚C before Illumina library preparation.