title: GSM8420565: XX_D2_H3K4me3_sgFoxd3_R3; Mus musculus; OTHER
library_construction_protocol: Following dissociation with Accutase, 1*105 cells per antibody were collectedand quickly washed in Wash buffer (20 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 10 mM Sodium butyrate, Protease Inhibitor, and 1 mM PMSF). 10 μl Concanavalin A beads (Bangs Laboratories) were equilibrated with 100 μl binding buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) and then concentrated in 10 μl binding buffer. The cells were bound to the Concanavalin A beads by incubating for 10 min at RT on a rotator. Following, the beads were separated using a magnet and resuspended in 100 μl chilled Antibody buffer (Wash buffer with 0.05% Digitonin and 2 mM EDTA). Subsequently, 1 μl of primary antibody (Supplemental Table S6) was added and incubated on a rotator for 3 hours at 4°C. After magnetic separation, the beads were resuspended in 100 μl chilled Dig-wash buffer (Wash buffer with 0.05% Digitonin) containing 1 μl secondary antibody and incubated for 1 hour at 4°C on a rotator. The beads were washed three times with chilled Dig-wash buffer and resuspended in chilled Dig-300 buffer (20 mM HEPES-KOH, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 10 mM Sodium butyrate, 1 mM PMSF) with 1:250 diluted 3xFLAG-pA-Tn5 preloaded with mosaic-end adapters. After incubation for 1 hour at 4°C on a rotator, the beads were washed four times with chilled Dig-300 buffer and resuspended in 50 μl Tagmentation buffer (Dig-300 buffer, 10 mM MgCl2). Tagmentation was performed for 1 hour at 37°C and stopped by adding 2.25 μl 0.5 M EDTA, 2.75 μl 10% SDS and 0.5 μl 20 mg/mL Proteinase K and vortexing for 5 s. DNA fragments were solubilized overnight at 55 °C followed by 30 min at 70 °C to inactivate residual Proteinase K. DNA fragments were purified with the ChIP DNA Clean & Concentrator kit (Zymo Research) and eluted with 25 μl elution buffer.10 μl Concanavalin A beads (Bangs Laboratories) were equilibrated with 100 μl binding buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) and then concentrated in 10 μl binding buffer. The cells were bound to the Concanavalin A beads by incubating for 10 min at RT on a rotator. Following, the beads were separated using a magnet and resuspended in 100 μl chilled Antibody buffer (Wash buffer with 0.05% Digitonin and 2 mM EDTA). Subsequently, 1 μl of primary antibody (Supplemental Table S6) was added and incubated on a rotator for 3 hours at 4°C. After magnetic separation, the beads were resuspended in 100 μl chilled Dig-wash buffer (Wash buffer with 0.05% Digitonin) containing 1 μl secondary antibody and incubated for 1 hour at 4°C on a rotator. The beads were washed three times with chilled Dig-wash buffer and resuspended in chilled Dig-300 buffer (20 mM HEPES-KOH, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 10 mM Sodium butyrate, 1 mM PMSF) with 1:250 diluted 3xFLAG-pA-Tn5 preloaded with mosaic-end adapters. After incubation for 1 hour at 4°C on a rotator, the beads were washed four times with chilled Dig-300 buffer and resuspended in 50 μl Tagmentation buffer (Dig-300 buffer, 10 mM MgCl2). Tagmentation was performed for 1 hour at 37°C and stopped by adding 2.25 μl 0.5 M EDTA, 2.75 μl 10% SDS and 0.5 μl 20 mg/mL Proteinase K and vortexing for 5 s. DNA fragments were solubilized overnight at 55 °C followed by 30 min at 70 °C to inactivate residual Proteinase K. DNA fragments were purified with the ChIP DNA Clean & Concentrator kit (Zymo Research) and eluted with 25 μl elution buffer.