GSM9450502: WT, replicate 1 (CUT&Tag); Mus musculus; OTHER
CUT&Tag was performed on sorted Treg cells form Foxp3YFP-CreKmt2dfl/fl(cKO) and Foxp3YFP-CreKmt2d+/+ mice to profile H3K4me3-enriched DNA fragments, following the NovoNGS CUT&Tag 4.0 High-Sensitivity Kit (for Illumina) (Cat. No.: N259-YH01; Novoprotein, Shanghai, China). Briefly, 1 × 106 Treg cells were harvested and incubated with NovoNGS ConA Beads for 10 minutes. The bead-bound cells were subsequently incubated overnight at 4°C with a primary antibody against H3K4me3 (Cell Signaling Technology), followed by a 1-hour incubation at room temperature with a secondary antibody while rotating. Tagmentation was initiated by adding a diluted ChiTag transposome and incubating for 1 hour at room temperature with rotation. Tagmentation Buffer was then added, and the mixture was incubated statically at 37°C for 1 hour. The reaction was terminated by adding Stop Buffer with vigorous vortexing, followed by a 10-minute incubation at 55°C. The resulting DNA libraries were purified using Tagment DNA Extract Beads; a two-volume equivalent of resuspended beads was added to the sample and incubated for 5 minutes at room temperature for DNA binding. The bead-bound DNA was captured magnetically, and the supernatant was discarded. The beads were washed twice with freshly prepared 80% ethanol. After air-drying, the purified DNA was eluted in Elution Buffer. These final library samples were undergoing PCR amplification to construct next-generation sequencing libraries, which were subsequently sequenced using the Illumina Novaseq X Plus platform. Two biological repeats were sequenced.