GSM9485396: WT_untreated_rep 1; Mus musculus; OTHER
Bone marrow cells were differentiated into macrophages in DMEM supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mM glutamine, 1× non-essential amino acids solution, 100 U/ml penicillin, 100 μg/ml streptomycin, and 20% (v/v) L929-conditioned medium at 37°C with 5% CO2. BMDMs were collected on day 5 and seeded for experiments overnight. HEK293T cells (ATCC CRL-3216, mycoplasma tested but not authenticated) were cultured in DMEM supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 1× non-essential amino acids solution, 100 U/ml penicillin and 100 μg/ml streptomycin. Reactions and Libraries were prepared with the CUTANA CUT&RUN and library prep kits from Epicypher (14-1048 and 14-1001), with slight modifications. BMDMs were fixed with 0.1% formaldehyde (ThermoFisher, 28908) for 1 minute, then quenched with glycine (CST, 7005S). Nuclei were isolated with CUTANA Nuclei Extraction Buffer (Epicypher, 21-1026). Nuclei were incubated with SPOP (Genentech, clone F5) or H3K27me3 (CST, 9733S) antibodies overnight. DNA was de-crosslinked by addition of 0.1% SDS and 0.23 μg/μL proteinase K (CST, 10012S), and incubation overnight at 55°C. Libraries were pooled and sequenced on NovaSeq 6000 (Illumina) to generate 20 million paired-end 50-base pair reads for each sample