GSM5777127: IFNg_STAT1 B (14994S) rep1; Mus musculus; OTHER
CUT&Tag was performed as previously described (Kaya-Okur et al., 2019) with minor adaptations. Primary OPCs were washed from the plate with PBS at room temperature. Cells were centrifuged for 5 minutes at 300xg and room temperature and then resuspended in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche Complete Protease Inhibitor tablet). 100 000 cells per condition were centrifuged for 5 minutes at 300xg and room temperature and resuspended in wash buffer. Activated Concanavalin-A beads (Bangs Laboratories BP531) in binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) was added to each condition and incubated for 10 minutes on a rotator at room temperature. Beads with nuclei were now kept on a magnetic stand and washed twice with Antibody buffer (2mM EDTA, 0.1% BSA and 0.05% digitonin in wash buffer). After discarding the liquid, beads were resuspended with primary antibody (STAT1, Cell Signaling 14994S; rabbit, monoclonal, 1:100) in antibody buffer and incubated overnight at 4°C on a rotator. Then beads were washed with freshly prepared Dig-wash buffer (0.05% digitonin in wash buffer) and incubated with secondary antibody (guinea pig anti-rabbit, NBP1-72763, Novus biologicals, 1:100) in Dig-wash buffer on a rotator for 30-60 minutes. After three washes with Dig-wash buffer, the beads were incubated with pA-Tn5  (Kaya-Okur et al., 2019) adapter complex (1:250) in Dig-300-wash buffer (300mM NaCl and 0.05% digitonin in wash buffer) and incubated for 1 hour at room temperature on a rotator. After two washes with Dig-300-wash buffer, the beads were resuspended in tagmentation buffer (10 mM MgCl2, 300mM NaCl and 0.05% digitonin in wash buffer) and incubated at 37° for 1 hr in a PCR cycler with heated lid. To stop the tagmentation reaction and to solubilize DNA fragments, 2.5 µL of 0.5M EDTA, 5 µL 10% SDS and 2 µL of 20 mg/ml Proteinase K was added to the beads and an incubation of 1 hour at 55°C. DNA from the samples was purified using the Zymo DNA Clean & Concentrator kit (D4014, Zymo) according to manufacturer's instructions, then amplified according to the number of cycles analyzed with qRT-PCR. SPRI bead (B23317, Beckman Coulter) purification was performed on the post-amplification libraries to remove remaining adapter dimers. Three replicates per condition were performed with primary OPCs obtained from different litters. Libraries were sequenced on the Illumina Nextseq 500/550 with 37-8-8-37 bp set-up. CUT&Tag (Kaya-Okur et al., 2019) . Libraries were sequenced on the Illumina Nextseq 500/550 with 37-8-8-37 bp set-up.