title: GSM8035703: Diff LUHMES, TG, input_A4; Homo sapiens; OTHER
library_construction_protocol: 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. All following steps were done in duplicate where each duplicate was a pool of 3 separate replicates. Per sample 100,000 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 primary antibody at a 1:50 dilution for both anti-H3K4me3 (Cell Signaling Technology C42D8) and anti-YY1 (Cell Signaling Technology D5D9Z). The following day, 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/ChIP-seq