title: GSM8491053: D3_Teff_Rest_high_IL2RA_screen; Homo sapiens; OTHER
library_construction_protocol: Cell sorting was performed 10 days following isolation for the resting screens. For the stimulated Teff screen, cells were restimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) 9 days following initial isolation and sorting was performed 72 hours after restimulation, at the time of peak IL2RA expression. Prior to sorting, cells were counted, washed once with EasySep buffer, and stained with Alexa Fluor® 647 anti-human IL2RA (CD25) Antibody (Biolegend, #302618). Cells were then washed and resuspended in EasySep buffer. During sorting, cells were gated on the GFP+ population (lentiviral guide library marker) and the top and bottom 20% of IL2RA expressing cells were sorted into 15 mL conical tubes coated with FCS. Isolated cells were pelleted, counted and lysed. gDNA extraction was performed using phenol-chloroform extractions. sgRNA was amplified and barcoded from the genomic DNA according to the protocol by Joung et al. (Joung et al. 2017). Up to 2.5 µg of genomic DNA were added to each 50 µL reaction, which included 25 µL of NEBNext Ultra II Q5 master mix (NEB, Cat #M0544L), 1.25 µL of the 10 µM forward primer and 1.25 µL of the 10 µM reverse primer, and H2O to 50 uL. The following PCR cycling conditions were used: 98°C for 3 minutes, followed by 23 cycles at 98°C for 10 seconds, 63°C for 10 seconds, and 72°C for 25 seconds, and ending with 2 minutes at 72°C. Samples were purified using 1X SPRI. The concentration of each sample was then measured using the Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific, Cat #Q32854). Libraries were sequenced on an Illumina HiSeq 4000 at the UCSF CAT using a custom sequencing primer.