title: CUT&RUN data of Nanog and Sox2 in mouse embryonic stem cells treated with auxin for CHD4 depletion against untreated control
design: CUT&RUN data of Nanog and Sox2 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 processed immediately. 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). MaxExtract tubes were spun down at 1500 x g for 1 minute at room temperature before use. Equal volume of Phenol/chloroform/isoamylalcohol (100µL) was added to each sample. Samples were vortexed for 15 seconds and then transferred into the MaxExtract tubes. Samples were spun down at 16,000 x g for 5 minutes at room temperature. 100µL of chloroform was added, tubes were inverted 10 minutes to mix and then spun down at 16,000 x g for 5 minutes at room temperature. The aqueous phase was transferred into new tubes were 2mg/mL glycogen and 500μl of 100% Ethanol were added. Samples were incubated at -20C for 30 minutes and then spun down at 20,817 x g for 15 minutes at 4C. The ethanol wash step was repeated. Ethanol was removed and samples were left to air-dry before being resuspended in 20µL of nuclease-free water. Library preparation for sequencing was carried out in the SCI facilities. 