Fitc anti rabbit secondary

Cells were washed once with PBS, then incubated in LIVE/DEAD™ Fixable Aqua Dead Cell Stain (Invitrogen L34965) diluted in PBS for 30 mins on ice. After a PBS wash, cells were fixed with 1.6% formaldehyde in PBS for 1 min, then quenched with an equal volume of 1X eBioscience permeabilization buffer (Thermofisher Scientific 00-8333-56). Cells were immediately centrifuged for 5 min at 600g and washed once with permeabilization buffer. Cells were then stained with anti-cleaved caspase 3-PE (BD #550821) and anti-GATA-1 (Abcam ab181544) for 30 mins at room temperature and FITC anti-rabbit secondary (Cell Signaling Technologies #4412S) for 30 mins. Washes were performed using permeabilization buffer and cells were resuspended in PBS for FACS and sorted using a BD FACSAria II. Cells positive for Aqua live/dead stain or cleaved caspase 3 were gated out and a narrow FSC gate was used to control for cell size. Cells in the lowest and highest 15% of GATA-1 expression were then sorted into PBS containing 30% FBS for InTAC-seq.