H3K27me3 and H3K4me3 Profiling by CUT&Tag

CUT&Tag was performed as in^{16 (link)} with minor modifications on 50,000 to 100,000 nuclei with indicated antibody (1/50, Cell Signaling Antibodies : Anti-H3K27me3, Ref: 9733- C36B11, Anti-H3K4me3, Ref: 9751- C42D8)^{16 (link),50} . All washes were performed in a volume of 500 µl and all centrifugations were done using a swinging bucket centrifuge at 1,300g, 4 min, at 4 °C for nuclei preparation and 600g, 8 min, 4 °C for subsequent steps. Nuclei were extracted and permeabilized from 10-20 mg frozen tumor tissues by incubating samples 10 min on ice in 6 ml ice-cold NE1 buffer (20 mM HEPES pH7.2, 10 mM KCl, 0.5 mM spermidine, 20% glycerol, 1%BSA, 1% NP-40, 0.01% digitonin, 1× proteases inhibitor) after mechanical dissociation. Following antibody incubation and tagmentation, samples were incubated for 1 h at 55 °C with max speed agitation with 3 µl 10% SDS and 2.5 µl 20 mg/ml proteinase K. After DNA extraction (Qiagen, Ref: 139046 MaXtract High density), PCR amplification (with 17 cycles, 20 s at 63 °C combined annealing/extension step) of the sequencing libraries was performed and profiles were checked on the Agilent TapeStation using High-sensitivity D1000 reagents. CUT&Tag libraries were sequenced on a NovaSeq 6000 (Illumina) in PE50 mode.