Cut and Run from Caki-1, MCF7, and U251 cell lines with NRF1 and CTCF
We performed a Cut & Run assay in the associated cell lines to profile the direct binding of NRF1 and CTCF  at the promoters of target genes. Overall design: Caki-1, MCF7, and U251 cell lines were cultured under designated conditions according to information on the American Type Culture Collection (ATCC) website (https://www.atcc.org/). When cells reached the desired confluence and numbers, the CUT&RUN Kit (14-1048, EpiCypher, USA) and CUT&RUN Library Prep Kit (14-1002, EpiCyher, USA) were applied following the manufacturer's protocols. Briefly, wash buffer, cell permeabilization buffer, and antibody buffer were freshly prepared on day 1. ConA Beads were activated by washing and then diluted with a cold bead activation buffer. Following these steps, 500,000 cells were harvested for each reaction, followed by resuspending in a wash buffer and mixing well with activated beads. After 10 minutes of incubation at room temperature, tubes were placed on an 8-strip magnet until slurries cleared. Supernatant was removed and a cold antibody buffer was added to each reaction. The SNAP-CUTANATMK-MetStat Panel was first added to the reactions designed for positive (H3K4me3) and negative (IgG) control antibodies. Then, 0.5 ug designated antibody was added to each reaction and incubated overnight. On the next day, antibody-bound histone PTM or chromatin-interacting protein were washed with the cell permeabilization buffer. Next, pAG-MNase was added to cleave target-DNA complexes. Targeted chromatins were then digested and released by adding calcium chloride, E. coli spike-in DNA, and Stop Buffer Master Mix. DNA was purified and up to 5ng CUT&RUN-enriched DNA was used for further library construction following the CUTANATM CUT&RUN Library Prep Kit. Library fragment sizes were analyzed using TapeStation and libraries were sequenced.