title: GSM9308654: Donor R29796, FACS sorted CD8+ CXCR3+ CM T cells, H3K27me3(Cut and Run); Homo sapiens; OTHER
library_construction_protocol: Of the FACS sorted CD8 T cells, 100,000 from each sorted population was collected for each respective immunoprecipitation pull down assay and an input sample. Some modifications were made to the manufacturer's protocol for the CUT & RUN Assay Kit (Cell Signaling Technologies, #86652). Briefly, live cells were washed (+spermine, +PIC) and mixed with activated Concanavalin A beads. After incubation, for IP samples: antibody binding buffer (+spermidine +PIC +digitonin) was added to clean cell:bead suspensions. Each reaction was incubated overnight with the respective antibody; anti-H3K4me3 (CST-C42D8), anti-H3K27me3 (CST-C36B11). Cells were washed and lysed with Digitonin Buffer (+spermidine, +PIC, +digitonin) and resuspended in the presence of pAG-MNase. Excess pAG-MNase was washed, and Calcium Chloride was added to activate the enzyme. The reaction was stopped with Stop Buffer (+digitonin, +RNase A, +spike-in DNA), and spun to collect the supernatant containing the fragmented DNA. Fragmented DNA was purified using Spin Columns (CST#14209) per manufacturer's protocol. The DNA fragments were repaired, A-tailed and further ligated with Illumina adapter. The final DNA library was obtained by size selection and PCR amplification.