GSM8078622: KO_CD4; Mus musculus; RNA-Seq
Thymi from 4 WT or PD-1 knockout animals were harvested and pooled. Thymi were disrupted with a 1-cc syringe plunger through a 70 um nylon cell strainer and filtered to obtain single-cell suspensions using FACS Buffer (PBS containing 2% FBS and 0.01% sodium azide). CD8+ cells were depleted using CD8a-biotin and rapid spheres followed by a second depletion with CD8b-biotin and rapid spheres, which resulted in enrichment of CD4+ thymocytes. Cells were stained with LIVE/DEAD fixable Aqua followed by extracellular staining with HSA, CD25, CD74, CD4, and CD8a. Cells were resuspended for sorting in FACS buffer and sorted on an Aria (BD Biosciences) using an 85-micron nozzle. Live cells were identified and then the following two populations were sorted: CD4+ HSA+ CD73- CD25- (CD4 single positive thymocytes) and CD4+ HSA+ CD73- CD25+ (regulatory T cells and CD25+ regulatory T cell progenitors) for RNA extraction. Library preparation was performed according to manufacturer's protocol (Next GEM Single Cell 5' Reagent Kit v2).