GSM5065940: WT IgG rep2 [CUT&Tag]; Mus musculus; OTHER
A hyperactive pA-Tn5 transposon for the Illumina platform was first generated. Cells were harvested and washed twice. Then the nuclei were isolated by ice-cold NE1 buffer and re-suspended in wash buffer. 10 μl of activated concanavalin A beads were added per sample and incubated at room temperature for 10 minutes. Primary antibody (1:50 dilution of anti-IgG, anti-H3K4me1, H34me3, H3K27me3 and H3K27ac) incubation was performed on a rotator at 4°C overnight. To increase the number of protein A binding sites, a secondary antibody (such as guinea pig anti-Rabbit IgG antibody for a rabbit primary antibody, and rabbit anti-mouse IgG for a mouse primary antibody) was diluted 1:100 in wash buffer and incubated at RT for 1h. After removing unbound antibodies, cells were mixed with 1:100 dilution of pA-Tn5 (0.04 µM) and incubated at room temperature for 1 hour on rotator. Next, cells were re-suspended in 300 µl tagmentation buffer and incubated at 37 °C for 1 h. To stop tagmentation, 10 µl 0.5 M EDTA, 3µl 10% SDS and 2.5 µl of 20 mg/mL Proteinase K was added to sample, which was incubated overnight at 37 °C. DNA was extracted by PCI and dissolved in 30 μl TE buffer. To amplify libraries, we added P5 primer, P7 primer (Vazyme, catalog. no. TD202), TruePrep Amplify Enzyme (Vazyme catalog. no. TD601) and performed PCR with the conditions below: 72°C for 3 minutes; 98°C for 30 seconds; 18 cycles of 98°C for 15 seconds, 63°C for 15 seconds; 72°C for 5 minutes. Post-PCR clean-up was performed by adding 1.2 volume of DNA clean beads (Vazyme, catalog. no. N411) and libraries were eluted in 20 μl nuclease-free water.