Facs aria

Peripheral blood mononuclear cells from healthy donors (Hoxworth blood center, Cincinnati, OH) were separated by centrifugation through Ficoll-Hypaque (GE, Fairfield, CT). CD4+ T cells were purified by negative selection using the Miltenyi CD4 negative separation kit (Auburn, CA), per the manufacturer’s instructions. Aliquots of purified CD4+ T cells were frozen in FBS + 10% DMSO and stored in liquid nitrogen. The viability of thawed cryopreserved cells was >90%. To isolate bulk Treg, cryopreserved CD4+ T cells were stained with live dead aqua (ThermoFisher, Waltham, MA), anti-CD8-FITC, anti-CD25-APC (BD Pharmingen, San Diego, CA), anti-CD127-PE (Beckman Coulter, Fullerton, CA), and sorted using a FACS Aria (BD) (supplemental Fig. S1A). Purity of the sorted Treg was >90%, as determined by postsorting analysis of FOXP3 expression (supplemental Fig. S1B). FOXP3 expression was also significantly higher in Treg compared with conventional CD4+T cells (non-Treg) after sorting (supplemental Fig. S1C). In some experiments, Treg subsets (nTreg, mTreg, and emTreg) were isolated, briefly cryopreserved CD4+ T cells were stained with anti-CD8-FITC, anti-CD25-APC, anti-HLA-DR-APC-Cy7 (BD), anti-CD127-PE (Beckman Coulter), anti-CD45RA-PB, anti-CD95-BV510 (BioLegend, San Diego, CA), and sorted using an FACS Aria (supplemental Fig. S1A).

A CRISPR/Cas9 approach was used to mutate the SELENOK gene (NCBI GeneID 58515) in order to generate a cell line expressing a truncated, nonfunctional SELENOK protein. First, SK-Mel28 cells were transfected with a pSpCase9(BB)-2A-GFP (Addgene) using Lipofectamine 3000 (ThermoFisher). After 24 h, GFP-positive cells were sorted using a FACSaria (BD Bioscience) into medium with 20% FBS without antibiotics. These cells were transfected with Integrated DNA Technologies (IDT) guide RNA and transactivating RNA complexes premixed in IDT complex buffer for 30 min using Lipofectamine 3000 per IDT recommendations. Cells were recovered in medium with 20% FBS without antibiotics for 24 hours, then 7-AAD Viability Stain Solution (Biolegend, Inc.) was added and another round of cell sorting carried out using the FACSaria instrument. The GFP⁺7AAD^- cells were sorted into 96-well plate with one cell per well in 200 mL of medium with 20% FBS and no antibiotics. After ∼2.5 weeks, live clones were transfered to 24-well plates and further grown into colonies. Colonies were screened by western blot for truncated SELENOK protein. Those clones exhibiting desired changes in SELENOK levels or size were confirmed as edited by Sanger sequencing.