GSM8728234: Treg FOXP3 low bin, Donor 2, CRISRPi screen; Homo sapiens; OTHER
Forty-eight hours post-restimulation, Tregs and Tconvs were counted, washed, and stained with Ghost Dye Red 780 Live/Dead stain (Tonbo, catalog no. 13-0865-T500) and antibodies targeting CD4 (Biolegend, catalog no. 344634), FOXP3 (Biolegend, catalog no. 320112) and HELIOS (Biolegend, catalog no. 137216). Cells were sorted on lymphocytes, singlets, live cells, BFP+ (a marker of the gRNA library plasmid), and FOXP3 high and low bins that captured the top and bottom ~25% of FOXP3 expression in each cell type. Following sorting, cells were pelleted and lysed, and genomic DNA (gDNA) was extracted using phenol-chloroform gDNA extraction. DNA regions containing the lentivirally integrated gRNA were amplified using PCR with custom primers. PCR reactions were conducted using 2 µg gDNA per 50 µL reaction with 0.5 µM forward and reverse primers, 0.075 U/µL TaKaRa Ex Taq DNA polymerase (Takara Bio, catalog no. RR001C), 0.2 mM dNTP, and 1X Ex Taq Buffer and amplified under the following cycling conditions: 1 min at 95°C, (30 s at 95°C, 30 s at 60°C, 30 s at 72°C) x 25 cycles, 10 min at 72°C, hold at 4°C. Following amplification, aliquots of individual samples were pooled, and primer dimers removed using a 1.4X SPRI bead cleanup. Proper amplification and concentration were assessed using Qubit quantification and DS1000 High Sensitivity Tapestation analysis, according to manufacturers' protocols. Samples were pooled equimolarly and sequenced on an Illumina HiSeq4000 instrument using a custom sequencing primer.