title: scRNA-seq CRISPR interference screen of CD4-positive, alpha-beta T cells, pooled from 2 donors, resting and 8 hr stimulation
design: Naive human CD4+ T cells were isolated isolated with the EasySep Human Naive CD4+ T Cell Isolation Kit (catalog no. #19555, STEMCELL Technologies) according to the manufacturers protocol from PBMC-enriched leukapheresis products (Leukopaks, STEMCELL Technologies, catalog no. 70500) from healthy donors, after institutional review boardapproved informed written consent (STEMCELL Technologies). After isolation (day 0), 30 million naive human CD4+ T cells were seeded at 1 x 106 cells/ml in X-VIVO 15 (Lonza Bioscience, catalog no. 04418Q) supplemented with 5% fetal calf serum (FCS) (cXVIVO) and 200 IU/ml of recombinant human IL-2 (Amerisource Bergen, catalog no. 10101641). Cells were activated using ImmunoCult human CD3/CD28/CD2 T cell activator (10990, STEMCELL Technologies) at 25 l/ml. The next morning, cells were transduced with 2% v/v concentrated ZIM3KRAB-dCas9-BlastR lentivirus. In the afternoon, cells were transduced with 0.1% v/v [multiplicity of infection (MOI) ~ 0.2] genome-scale sgRNA library lentivirus. At day 3, cells were centrifuged and resuspended with above medium supplemented with 200 IU/mL IL-2. Blasticidin and puromycin were added to 10 g/mL and 2 g/mL final concentrations, respectively. At day 5, cells were counted and diluted to 5 x 105 cells/mL with cXVIVO supplemented with 200 IU/mL IL-2 and 10 g/mL blasticidin. Recombinant human IL-7 (R&D Systems, catalog no. 207-IL) was also added to a final concentration of 5 ng/mL. At day 7, cells were counted and diluted to 7.5 x 105 cells/mL with cXVIVO supplemented with 200 IU/mL IL-2, 5 ng/mL IL-7 and 10 g/mL blasticidin. At both day 8 and day 10, cells were centrifuged and resuspended at 5 x 105 cells/mL with cXVIVO supplemented with 200 IU/mL IL-2 and 5 ng/mL IL-7. At day 12 morning, cells were centrifuged and resuspended at 1 x 106 cells/ml with cXVIVO supplemented with 200 IU/mL IL-2 and split into three populations. The first population (Rest) was kept at a resting state without restimulation and harvested in the afternoon after 8 hours. The second population (Stim8hr) was restimulated by adding 12.5 L/ml ImmunoCult human CD3/CD28/CD2 T cell activator and harvested in the afternoon after 8 hours. The third population (Stim48hr) was restimulated by adding 12.5 L/ml ImmunoCult human CD3/CD28/CD2 T cell activator and harvested after 48 hours. Cells were harvested, fixed and stored using GEM-X Flex Sample Preparation v2 Kit (10x genomics, catalog no. 1000781) per the manufacturers instructions.