GSM8728226: Treg FOXP3 low bin, Donor 2, CRISRP KO TF screen; Homo sapiens; OTHER
Forty-eight hours post-restimulation, cells were counted and stained with Ghost Dye Red 780 Live/Dead stain (Tonbo, catalog no. 13-0865-T500) and antibodies targeting FOXP3 (Biolegend, catalog no. 320116) and HELIOS (Biolegend, catalog no. 137216). Cells were sorted on lymphocytes, singlets (x2), live cells, GFP+ (a marker of the gRNA library plasmid), and FOXP3 high and low bins that captured the top and bottom 25% of FOXP3 expression. Following sorting, cells were pelleted and lysed, and 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 1.75 µg gDNA per 50 µL reaction with 0.25 µM forward and reverse primers and 1X NEBNext Ultra II Q5 Master Mix (New England Biolabs, catalog no. M0544). Reactions were amplified under the following cycling conditions: 3 min at 98°C, (10 s at 98°C, 10 s at 63°C, 25 s at 72°C) x 23 cycles, 2 min at 72°C, hold at 4°C. Following amplification, aliquots of individual samples were pooled, and primer dimers removed using a 1.25X 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.