GSM8450323: Valid_EB_CT-EB_ZbHA_H3K4me3-d3-CUT&Tag 2; Mus musculus; OTHER
Cut&Tag was performed as previously described using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme Biotech, TD904). Briefly, cells were bound to 10μL of pre-washed ConA beads in a 200μL low-binding tube and incubated overnight at 4°C with 50 μL of antibody buffer containing 0.5μg of the target antibody. After two washes with dig-wash buffer, cells were incubated with 50μL of dig-wash buffer containing 0.5μg of a secondary antibody for 1 hour at room temperature. Following two additional washes with 200μL of dig-wash buffer, tagmentation was performed by adding 1μL of pG-Tn5 in 100μL of dig-300 buffer and incubating for 1 hour at room temperature. After two final washes with 200μL of dig-wash buffer, tagmentation buffer was added, and samples were incubated at 37°C for 1 hour. The reaction was stopped by adding 10% SDS, and tagmented DNA fragments were purified using VAHTS beads. Following purification, libraries were amplified by PCR. Sequencing libraries were prepared continue using the Hyperactive In-Situ ChIP Library Prep Kit (Vazyme Biotech, TD904). The index used for library preparation were from the TruePrep index Kit V2 for Illumina (Vazyme Biotech, TD202)