GSM7574434: B16_WT_H3K4me3 CUT&Tag; Mus musculus; OTHER
The CUT&Tag assay was conducted using Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903). Briefly, 5 × 104 B16 cells were collected and washed once with 500 μl wash buffer before they were bound to activated ConA beads for 10 min at room temperature. After that, cells were incubated with 1 μg anti- H3K9me3 (CST#13969), H3K4me3 (ab8580) or p300 (active motif 61401) antibody at 4°C overnight, respectively. Anti-rabbit IgG was added and incubated for 1 h at 25°C the next day. Then cells were washed three times with DIG wash buffer and incubated with 0.04 μM pA/G-Tnp for 1 h at 25°C. Similarly, cells were washed three times with DIG 300 buffer, resuspended in tagmentation buffer and incubated at 37°C for 1 h. Tagmentation was stopped by adding proteinase K, buffer LB and DNA extract beads. After incubation at 55°C for 10 min, cells were plated on a magnet and unbound liquid was removed. Beads were gently rinsed twice with 80% ethanol and DNA was eluted with double-distilled water. Libraries were constructed using TD202 TruePrep Index Kit V2 for Illumina from Vazyme Biotech. For library amplification, 15 μl of purifed DNA was mixed with 25 μl of 2 × CAM, along with 5 μl of uniquely barcoded i5 and i7 primers from the TruePrep Index Kit V2 for Illumina (Vazyme Biotech, TD202). A total volume of 50 μl of sample was placed in the Mastercycler nexus (Eppendorf, 6331000076) using the following program: 72°C for 3 min; 98°C for 3 min; 18 cycles of 98°C for 10 s, 60°C for 5 s, and 72°C for 1 min; and holding at 12°C. To purify the PCR products, 2 × volumes of VAHTS DNA Clean Beads (Vazyme Biotech, N411) were added and incubated at room temperature for 5 min. The beads were washed twice with 200 μl fresh 80% ethanol and eluted in 22 μl ddH2O. All CUT&Tag libraries were sequenced using the Illumina NovaSeq 6000 platform in PE150 mode.