title: GSM7790931: NES, EV, V5 CUT&RUN, Replicate 2; Homo sapiens; OTHER
library_construction_protocol: Preparation of CUT&RUN libraries was done with the CUT&RUN assay kit (Cell Signaling Technology). Briefly, 250,000 NES cells per sample were fixed with 0.1% formaldehyde in PBS for 2 min and quenched with 1 M glycine for 5 min at room temperature. Cells were washed in 1x wash buffer containing spermidine and protease inhibitor cocktail, and incubated in antibody binding buffer containing spermidine, proteinase inhibitor cocktail, digitonin, and 10 µl of activated concanavalin A beads per sample for 16-18 h at 4 C. Per CUT&RUN reaction, 0.2 µg of H3K27me3 antibody (clone C36B11, #9733, Cell Signaling Technology) or 0.96 µg of V5 antibody (clone D3H8Q, #13202, Cell Signaling Technology) was used. pAG-MNase in 1x wash buffer containing spermidine, protease inhibitor cocktail, and digitonin was bound to the antibody for 1 h at 4 C. pAG-MNase was activated by adding 3 µL calcium chloride per sample and incubated for 30 min at 4 C. Digestion was stopped by adding 1x stop buffer (digitonin solution, RNase A, and 50 pg S. cerevisiae spike-in DNA (Cell Signaling Technology) for normalization) and samples were incubated for 10 min at 37 C. Samples were subjected to SDS and proteinase K treatment for 2 h at 65 C. Zymo DNA Clean and Concentrator (D4034) was used to clean up the samples. NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645L) was used for library preparation. Library clean-up and size selection was done by using SPRI beads (103778-488, Bulldog Bio). Libraries were quantified and analyzed by Qubit and Tapestation 4200 (Agilent) analysis and sequenced for 38 cycles in paired-end mode on a NextSeq 500 (Illumina).