GSM8207699: T Cells, CHRONIC exhaustion, H3K27me3, rep R2; Mus musculus; OTHER
The CUT&RUN assay was performed as previously described with some modifications. In brief, after sorting for live CD8 positive T cells from the in vitro model, 20,000 cells per antibody were washed twice with cell wash buffer, bound to 40 ul of concanavalin A-coated magnetic beads, and then split to incubate overnight rotating at 4 °C in 250 ul of antibody buffer with either H3K27me3 (Cell Signaling, #9733) or H3K27ac (Active Motif, #39133) antibodies. Cells were then washed and resuspended in 0.1% digitonin buffer with pA-MN fusion protein at 700 ng/ml and incubated rotating at 4 °C for 1 hour. Cells were then washed, resuspended in the digitonin buffer, brought down to 0 °C, and CaCl2 was added to a final concentration of 2mM for digestion at 0 °C for 30 minutes. 2X stop buffer was then added to the reaction and incubated at 37°C for 10 minutes to release the fragments followed by centrifugation. SDS (0.1%) and proteinase K (0.2 mg/ml) were then added to the supernatant, incubated at 70°C for 10 minutes, and followed by phenol-chloroform DNA extraction. Library preparation was performed according to manufacturer's guidelines using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645L). The NEBNext® Library Quant Kit for Illumina® (E7630L) and bioanalyzer were used to assess library quality and concentration. Libraries were sequenced using the Illumina® NextSeq 500.