GSM8488411: Diff LUHMES, TG, BG4+FLAG; Homo sapiens; OTHER
Differentiated LUHMES cells were treated with 10 nM TG or DMSO and collected 24 hours later. Cells were lifted via trypsinization, and triplicate conditions were mixed and counted.  Per sample 250,000 cells were aliquoted and washed two times using 1x wash buffer (+ spermidine and protease inhibitor cocktail). Cells were then immobilized with concanavalin A beads and incubated overnight at 4ºC with the BG4 antibody (MABE917) at a 1:50 dilution. The next day, the primary BG4 antibody was removed, cells were washed once using digitonin wash buffer, and the anti-FLAG secondary antibody (Sigma Aldrich F1804) was added at a 1:100 dilution for 1 hour at room temperature. Control samples that were not incubated with the BG4 primary received anti-FLAG only. Following wash steps after anti-FLAG incubation, pAG-MNase mixture was added and incubated at 4ºC for 1 hour. After 3 washes with digitonin wash buffer cells were cooled on ice for 5 minutes and pAG-MNase was activated with cold calcium chloride and incubated at 4ºC for 30-minute. 1x stop buffer was added and diffused DNA-samples were purified using DNA spin columns. Input samples were prepared via sonication using 6x 15 seconds pulses with 30 seconds pauses. Input fragment size of ~300-400 bp was confirmed using agarose gel electrophoresis. Enriched DNA samples were then submitted for library prep and paired-end sequencing done by Novogene (Sacramento, CA). Novogene library prep for CUT&RUN