title: GSM8240958: 10A_ev_dox, YAP1, rep3; Homo sapiens; OTHER
library_construction_protocol: CUT and RUN experiments were performed as described previously (Kim et al., Nat. Commun., 2022). Briefly, for each CUT and RUN reaction 200,000 cells were trypsinized, washed, resuspended in 100 µl wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM Spermidine) and bound to 10 µl activated BioMag®Plus Concanavalin A magnetic beads (Polysciences) for 10 min at room temperature. The cells were then incubated with antibodies diluted in 100 µl antibody buffer (wash buffer + 0.01% digitonine and 2 mM EDTA) overnight at 4°C. IgG rabbit antibody was used as negative control. After incubation with antibodies, the beads were washed in digitonin wash buffer (wash buffer + 0.01% digitonin) and incubated 1h at 4°C with 1 µg/ml protein A/G Micrococcal Nuclease fusion protein (pA/G MNase). After washing with digitonin wash buffer, beads were rinsed with low salt buffer (20 mM HEPES, pH7.5, 0.5 mM Spermidine, 0.01% digitonine), resuspended in 200 µl incubation buffer (20 mM HEPES, pH7.5, 10 mM CaCl2, 0.01% digitonin) and placed at 0°C to initiate cleavage. After 30 min, reactions were stopped by adding 200 µl STOP buffer (170 mM NaCl, 20 mM EGTA, 0.01% digitonin, 50 µg/ml RNAse A) and the samples were incubated 30 min at 37°C to digest the RNA and release the DNA fragments. The samples were then treated with proteinase K for 1 h at 50°C and the DNA was purified using Phenol/Chloroform/Isoamyl alcohol. After precipitation with glycogen and Ethanol, the DNA pellet was resuspended in 0.1 X TE and used for library preparation. For DNA library generation, the NEBNext®Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs) was used. Adaptor ligation was performed with 1:25 diluted adaptor and 15 cycles were used for library amplification using dual indices (NEB dual index kit). Paired-end 2x25 bp sequencing was performed.