GSM6927648: K562-CAS-EZH2i-IFN-H3K4me3; Homo sapiens; OTHER
Nuclei were isoalted by permeabilisting cells in NE1 Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl 0.5 mM spermidine, 0.1% Triton-X100, 20% glycerol, 1x Roche complete EDTA-free protease inhibitor) on ice for 10 minutes. Nuclei were resuspended in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1 x Roche complete EDTA-free protease inhibitor). Nuclei were bound to activated Concanavalin A beads. Bead-bound nuclei were resuspended in Anitbody Buffer (Wash Buffer supplemented with 2 mM EDTA and 0.1% BSA) and incubated with the antibody for 2h at room temperature. Samples were washed in 300 Buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1 x protease inhibitor cocktail) then incubated in 300 Buffer with CUTANA pAG-Tn5 enzyme (EpiCypher) for 1h at room temperature. Samples were washed. Then Incubated in Tagmentation Buffer  (300 Buffer supplemented with 10 mM MgCl2) for 1h at 37ºC. Tagmentation was stopped by STOP Buffer (300 Buffer supplemented with 40 mM EDTA, 0.2% SDS, 400 μg/mL proteinase K) and incubated at 55ºC for 2h, then held at 4ºC overnight. DNA was purified with MinElute PCR purification Kit (Qiagen). To amplify libraries, 21 μL of eluted DNA was mixed with 2 μL of a universal i5 primer, 2 μL of a uniquely barcoded i7 primer (Buenrostro, J.D. et al., Nature, 2015) and 25 μL of NEBNext HiFi 2X PCR Master Mix. The samples were amplified using the following cycling conditions: 72°C for 5 min (gap filling); 98°C for 30 s; 14 cycles of 98°C for 10 s and 63°C for 10 s; final extension at 72°C for 1 min and hold at 8 °C.