title: GSM8368464: D1-Teff-Stim-AAVS1-KO-H3K4me3_CUT&RUN; Homo sapiens; OTHER
library_construction_protocol: CUT&RUN from resting and 48-hour restimulated cells was performed according to the manufacturer's protocol with the EpiCypher CUTANA™ ChIC/CUT&RUN Kit and provided reagents. In brief, 5E5 T cells per reaction were washed with PBS before nuclear isolation using the EpiCypher recommended lysis buffer consisting of 20 mM HEPES pH 7.9 (Sigma-Aldrich), 10 mM KCl (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich), 20% Glycerol (Sigma-Aldrich), 1 mM MnCl2 (Sigma-Aldrich), 1X cOmplete Mini-Tablet (Roche, # 11873580001), and 0.5 mM Spermidine (Sigma-Aldrich). The cells were resuspended in 100 µL per reaction cold nuclear extraction buffer and incubated on ice for 10 minutes. Following lysis, nuclei were pelleted and resuspended in 100 µL per reaction of nuclear extraction buffer. The isolated nuclei were then frozen at -80 in extraction buffer until DNA isolation. After thawing the samples at 37C, the nuclei were bound to activated conA beads. After adsorption of nuclei to beads, permeabilization was performed with 0.01% digitonin containing buffer. Antibodies for H3K4me1 (EpiCypher, #13-0057), H3K4me2 (EpiCypher, #13-0027), H3K4me3 (EpiCypher, #13-0041) and IgG (EpiCypher, #13-0042) were added at 500 ng per reaction. Following overnight antibody binding, pAG-MNase addition, and chromatin cleavage, 0.5 ng of the provided E. coli DNA was added to each sample following chromatin cleavage by MNase. The provided spin columns and buffers were used for DNA isolation and purification. The resulting DNA was prepared for sequencing using the CUTANA™ CUT&RUN Library Prep Kit (Cat 14-1002) according to the manufacturer's protocol.