title: GSM6927855: KO-1; Mus musculus; OTHER
library_construction_protocol: The CUT&Tag assay was conducted following the protocol of Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, TD903) Briefly, 250 GV oocytes 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 antibody (Active Motif, # 39161) at 4°C overnight. 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. 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.