GSM8943324: IgG CUT&Tag control; Mus musculus; OTHER
Splenic CD8+ T cells were suspended in ice-cold PBS and washed for two times with wash buffer before combining with ConA beads at room temperature for 10 min. Binding sites of H3K27me3 DNA libraries were prepared by Hyperactive Universal CUT&Tag Assay Kit for Illumina (TD903, Vazyme). Briefly, a total of 1×105 splenic CD8+ T cells were suspended in ice-cold PBS and washed for two times with wash buffer before combining with ConA beads at room temperature for 10 min. Subsequently, 1 μg primary antibodies (anti-H3K27me3) were incubated with cell-ConA beads compound at 4 ℃ overnight. 0.5 μg secondary antibodies were added and incubated for 1 h at room temperature, and then washed for three times by Dig-wash buffer. Thereafter, cells were incubated with pA/G-Tnp transposon for 1 h and fragmented in tagmentation buffer for 1 h at 37 ℃. The fragmentation was stopped by stop buffer (proteinase K, Buffer L/B and DNA extract Beads) at 55 ℃ for 10 min. The stopped samples were placed on magnetic stand to extract DNA and amplified utilizing TruePrep Index Kit V2 for Illumina (TD202, Vazyme). The amplified DNA products were purified by VAHTS DNA Clean Beads (N411, Vazyme) for the final DNA libraries. The prepared DNA libraries were sequenced by Novaseq-PE150 platform at NovoGene (Beijing, China).