title: GSM8762495: SETDB1 KO control rep1; Mus musculus; OTHER
library_construction_protocol: Briefly, after harvesting, CD25+ enriched T cells were counted and divided into groups of 200,000 cells each. The cells were centrifuged at 600 g for 5 minutes at room temperature. Each sample was resuspended in 100 μl of pre-cooled NE Buffer, mixed gently, and incubated on ice. Following incubation, the cells were centrifuged again at 600 g for 5 minutes at room temperature, and the pellets were resuspended in 100 μl of wash buffer. Next, 10 μl of Concanavalin A-coated magnetic beads, pre-activated at room temperature, were added to each sample and incubated for 10 minutes. The unbound supernatant was removed, followed by an overnight reaction between the H3K9me3 group sample and anti-H3K9me3 antibodies (1:50, Proteintech), as well as between the SETDB1 group sample and anti-SETDB1 antibodies (2 μg, Proteintech) in Antibody Buffer at 4°C. After washing, the bound antibodies were reacted with secondary antibodies and then incubated with pA/G-Tnp Pro (0.04 μM). Next, the samples were fragmented with TTBL, followed by the addition of 10% SDS and DNA spike-in to each sample. To extract the genomic DNA, DNA Extract Beads Pro were activated and added to each sample. The extracted DNA fragments were amplified by PCR with indexing primers (Vazyme, TD202), and the library was purified using VAHTS DNA Clean Beads (Vazyme, N411).