GSM8840957: CD8 T cell GFP low donor 2; Homo sapiens; OTHER
Retroviral library was produced by transfecting 4.5 ug of the library plasmid DNA into FLYRD18 retroviral packaging cells, as described above. HEK and HeLa cells were cultured to a density of 60% prior to addition of virus-containing supernatants, and subsequently expanded for 10 days. PBMCs were activated with αCD3/ αCD28 and spinfected with virus-containing supernatants, as described above, and expanded for 10 days in RPMI supplemented with 10% fetal bovine serum (FBS; Bodinco), 2 mM L-glutamine, 20 IU/mL penicillin G sodium salts, 20 μg/mL streptomycin sulfate (all Sigma Aldrich), Non-Essential Amino Acids (Gibco), Sodium pyruvate (Gibco), 50 IU/mL recombinant human (rh) IL-2 (Proleukin, Novartis), 5 ng/mL rhIL-15 (Peprotech), and 10 ng/mL rhIL7 (Miltenyi). Transduction efficiencies were kept between 5-10% in order to minimize multiple transgene integrations. After expansion cells were harvested and stained with Near-IR live/dead marker (Life Technologies) for dead cell exclusion, in addition to anti-CD8-AF700 (Biolegend) and anti-CD4-BV510 (Biolegend) in the case of the T cell assay. The top and bottom 15% GFP expressing cells, in addition to the full GFP-positive fraction, were sorted on a FACS Aria II (BD Biosciences). The sorted cells were subsequently pelleted and resuspended in 1 µl DirectPCR Lysis Reagent (Viagen) per 2,500 cells, supplemented with 0.5 mg/ml proteinase K (Viagen). The obtained lysate was incubated at 55 C for 3 hours, followed by 85 C for 45 minutes, and 95 C for 5 minutes. An initial pre-amplification of the synthetic 3'UTRs was performed on 10 µl lysate (±50,000 cells) in a 50 µl reaction volume using the NEBnext (NEB) and 10 µM forward (5'-AAAGACCCCAACGAGAAGC-3') and reverse (5'-AGTCTATAGCTACTAGGCG-3') primers. An initial touchdown stage was performed dropping the annealing temperature 1°C per cycle from 58°C to 50°C (8 cycles), followed by 6 cycles at 50°C. Extension time was kept at 20 seconds. PCR products were purified using Cytiva Sera-Mag™ Select at a 1.2:1 Sera-Mag:PCR-product ratio, following manufacturers protocol. Each of these samples were subsequently used as template for a second PCR, with primers (Forward: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGACCCCAACGAGAAGCGCGATCAC-3', Reverse: 5'-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGTCTATAGCTACTAGGCGATA-3') designed to amplify the product and attach partial Illumina adapters. This second PCR was performed using the NEBnext (NEB) and consisted of 10 cycles with a constant annealing temperature of 65 °C and extension time of 30 sec. Correct products (±275 base pairs) were purified using Cytiva Sera-Mag™ Select at a 1.2:1 Sera-Mag:PCR-product ratio, following manufacturers protocol. Purified products were sequenced at the genomics core facility of the Netherlands Cancer institute.