DonorH cells perturbed with HEXDC knockdown by CRISPRi in batch Rep1 collected 48 hrs after restimulation
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). At day 0, primary naive human CD4+ T cells were seeded at 1 x 106 cells/ml in cXVIVO and 200 IU/ml IL-2 at 150L/well in 96-well flat bottom plates. Cells were activated using ImmunoCult human CD3/CD28/CD2 T cell activator at 25 l/ml. The next morning, cells were transduced with 2% v/v concentrated ZIM3KRAB-dCas9-BlastR lentivirus. In the afternoon, cells in each well were transduced with a distinct, individual single gRNA expressing lentivirus at 2% v/v. On day 3, cells were split to ~5 x 105 cells/mL and Blasticidin was added at 10 g/mL final concentration. At day 5, cells were centrifuged and resuspended with cXVIVO supplemented with 200 IU/mL IL-2, 5 ng/mL IL-7 and 10 g/mL blasticidin at ~5 x 105 cells/mL. At day 7, cells were counted and diluted to 1 x 106 cells/mL with cXVIVO supplemented with 200 IU/mL IL-2, 5 ng/mL IL-7 and 10 g/mL blasticidin. At day 8, cells were centrifuged and resuspended at 1 x 106 cells/mL with cXVIVO supplemented with 200 IU/mL IL-2 and 5 ng/mL IL-7. On day 10 morning, cells were centrifuged and resuspended at 2 x 106 cells/ml with cXVIVO supplemented with 200 IU/mL IL-2 and 12.5 L/mL ImmunoCult human CD3/CD28/CD2 T cell activator for 8 hours. Cells were centrifuged and resuspended in DNA/RNA Shield (Zymo Research) and sent for bulk RNAseq by Plasmidsaurus.
SUB16215586
Tact_cluster_validation_Diff092_DonorH_Stim48hr_Rep1_HEXDC_lib