Flow cytometry

Confluent 10 cm plates of mESCs were treated with 10 μM of EdU and incubated at 37^o C for 30 min. Cells were then washed with PBS and trypsinized. After spinning down, cell pellets were washed once with PBS then fixed by resuspending in a solution of 500 μL PBS and 4% PFA (paraformaldehyde) and incubating for 15 min at room temperature. Cells were washed twice with 1 mL 1% BSA in PBS. Cells were then resuspended in 1 mL 1% BSA + 0.5% Triton X-100. Cells were incubated at room temperature for 15 min then pelleted. Cells were suspended in a labeling solution of PBS, 1 mM CuSO₄, ~ 1 μM AlexaFlour 647 Azide, and 100 mM ascorbic acid. Cells were incubated at room temperature for 30 min, protected from light. 1 mL 1% BSA + 0.5% Triton X-100 was added before pelleting cells. Cells were resuspended in 1% BSA + 0.5% Triton X-100. To one biological replicate, 1 drop FxCycle Violet (Thermo Fisher, {"type":"entrez-nucleotide","attrs":{"text":"R37166","term_id":"794622","term_text":"R37166"}}R37166) reagent was added to each sample before sorting with Attune Nxt flow cytometer (Life Technologies). For two additional biological replicates, cells were resuspended in a solution of 100 μg/mL RNAse and 1 μg/mL DAPI and incubated at 37 ^o C for one hour before samples were sorted on Attune NxT flow cytometer. Cell cycle analysis was performed using FCS Express 7.0 software (De Novo, Glendale, CA). Total number of replicates including biological and technical is n = 5 for WT and n = 6 for Wiz^del cells.