GSM6627984: CUT&Tag_FOXO1_H1_4C; Homo sapiens; OTHER
The cells were lysed  by gentle pipetting and placed on ice for 10 min. The cell nuclei remained intact by this treatment, and were collected by centrifuged for 5 min at 500 g at 4°C. The nucleus fraction were resuspended by 200 μl PBS, cross-linked by adding 2 μl of 10% Formaldehyde and incubated at room temperature for 2 min. The cross-link reaction was terminated by adding 5 μl of 25 mM glycine. The cross-linked cell nuclei were centrifuged for 5 min at 600 g at room temperature. The pellets were resuspended in 90 μl wash buffer. Then, 10 μl of Concanavalin A-coated magnetic beads were equilibrated at room temperature and added into each sample, incubation continued for 15 min. The unbound supernatant was removed and bead-bound cells were resuspended in a 1:50 dilution of FOXO1 primary antibody. Primary antibody incubation was performed on a rotating platform at 4°C overnight. Followed by incubation with a secondary antibody at room temperature for 1 h to increase the number of IgG molecules at each epitope bound by the primary antibody. Cell nuclei were washed and incubated with pA-Tn5 adapter complex at room temperature for 1 h and washed under stringent conditions following manufacturer's instruction. Next, cell nuclei were resuspended in 50 μl Tagmentation buffer by addition of Mg2+ and incubated at 37°C for 1 h. To stop tagmentation, 1ul 10% SDS was added to 50 μl of sample, which was incubated overnight at 37 °C. To extract the genomic DNA, Tagment DNA Extract Beads were added to each tube. Samples were enriched by PCR amplification and a single solid-phase reversible immobilization (SPRI) magnetic bead cleanup step. For the negative control, we omitted the primary antibody, while the secondary antibody could randomly coat the chromatin at low efficiency without sequence bias. The purified DNA was used for library preparation. library quality was assessed on the Agilent Bioanalyzer 2100 system, and pair-end sequencing was performed on an Illumina platform (Novogene, Beijing, China).