GSM7720230: 3T3_H2AK119Ub_C3_rep1; Mus musculus; OTHER
CUT&Tag experiments were performed as previously described with modifications (H. S. Kaya-Okur et al., 2019.). Briefly, cells suspended in washing buffer were Concanavalin A coated magnetic beads (Polyscience, 86057) for 10 min at room temperature. Cells were then incubated with primary antibody (H3K4me3, Active motif #39160; H3K27me3, Cell Signaling Technology 9733 C36B11; H2AK119ub, Cell Signaling Technology 8240s; H3K9me3, active motif #39161) at a ratio of 1:100 at 4°C for 2 h on a thermomixer at 400 rpm. After the removal of primary antibody, secondary antibody (Invitrogen, #SA5-10228) were added at a ratio of 1:100, and incubation was performed for 1 h at 4°C. After washing two times, beads were incubated with protein A-Tn5 (Vazyme, S603) at 4°C for 1 h. Tagmentation was performed at 37°C for 30 min after washing. SDS was then added to stripe Tn5 and tagmented DNA was purified with 3 × VAHTS DNA Clean Beads (Vazyme, N411). PCR was performed to amplify the library for 16 cycles using the following PCR conditions: 72 °C for 3 min; 98 °C for 30s; and thermocycling at 98°C for 15s, 60°C for 30s and 72°C for 3min; followed by 72 °C 5 min. After the PCR reaction, libraries were purified with the 0.4x-1.7x AMPure (Beckman) beads size selection and were subjected to next generation sequencing.