title: CUT&Tag data of MBD3 in mouse embryonic stem cells treated with auxin for CHD4 depletion against untreated control
design: CUT&Tag data of MBD3 in mouse embryonic stem cells treated with auxin for CHD4 depletion against untreated control
library_construction_protocol: Cells were cultured on 10cm plates. Cells from all conditions were harvested at the same time using accutase and spun down for 3 min, 600 x g in a swinging bucket rotor at room temperature. Cells were washed in PBS and then resuspended in NE1 buffer (20mM HEPES-KOH pH7.9, 10mM KCl, 0.5mM spermidine, 0.1% Triton X-100, 5% Glycerol, protease inhibitors). They were incubated on ice for 10min. Nuclei was washed with PBS and then resuspended in wash buffer (20mM HEPES pH7.5, 150mM NaCl, 0.05mM Spermidine, 0.005% Igepal CA-630) with 10% DMSO. Nuclei was frozen down in Mr Frosty containers overnight. ES cells were cultured on 0.1% gelatin-coated plates in 2i/LIF conditions. For CHD4-mAID depletion, 0.5mM auxin (1:1000 from DMSO stock) was resuspended in pre-warmed culture media and transferred to the cells. Control cells were not treated with auxin (no auxin). After PCR amplification, samples were purified using SPRI beads at a ratio of 1.3× the PCR reaction volume (65 µL beads per 50 µL PCR product). Beads were mixed thoroughly with the PCR products by pipetting and incubated at room temperature for 5–10 min, followed by magnetic separation until the solution cleared. The supernatant was discarded, and the beads were washed twice with 200 µL of 80% ethanol while on the magnetic stand. After a brief centrifugation, residual ethanol was carefully removed without allowing the beads to over-dry. DNA was eluted from the beads by resuspension in 22 µL of 10 mM Tris-HCl (pH 8.0), incubated at room temperature for 60 min. For PCR amplification and indexing, 15 µL of 6% Triton neutralisation solution, 2 µL of 10 µM universal or barcoded i5 primer, and 2 µL of 10 µM uniquely barcoded i7 primer (a distinct barcode for each sample) were added to the bead slurry after Tn5 tagmentation. Tubes were vortexed briefly at full speed, centrifuged, and placed on a magnetic stand to separate the beads. The supernatant was transferred to a fresh tube, mixed with 25 µL of NEBNext polymerase (non–hot-start), vortexed, and briefly centrifuged. Samples were immediately placed in a thermocycler with a heated lid, using the following programme: 58 °C for 5 min, 72 °C for 5 min, 98 °C for 5 min, followed by 11 cycles of 98 °C for 10 s and 60 °C for 10 s, then 72 °C for 1 min, and a final hold at 8 °C.