GSM8457393: CD4 CROP-seq well12; Homo sapiens; RNA-Seq
Primary human CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic negative selection using the EasySep Human CD4+ T Cell Isolation Kit (STEMCELL, Cat #17952). Cells were cultured in X-Vivo media, consisting of X-Vivo15 medium (Lonza, Cat #04- 418Q) with 5\% Fetal Calf Serum, 50mM 2-mercaptoethanol, and 10mM N-Acetyl L-Cysteine. On the day of isolation (Day 1), cells were rested in media without stimulation for 24 hours. The day after isolation (Day 2), cells were stimulated with ImmunoCult Human CD3/CD28 T Cell Activator (STEMCELL, Cat #10971) and IL-2 at 50U/mL. 24 hours post stimulation (Day 3), 1 uL of lentivirus was added directly to cultured T cells and gently mixed. Following 24 hours (Day 4), cells were collected, pelleted, and washed in PBS twice. Then, cells were resuspended in Lonza electroporation buffer P3 (Lonza, Cat #V4XP-3032). Cas9 protein (MacroLab, Berkeley, 40mM stock) was added to the cell suspension at a 1:10 v/v ratio. Cells were transferred to a 96 well electroporation cuvette plate (Lonza, cat #VVPA-1002) for nucleofection using the Lonza Nucleofector 96-well Shuttle System and pulse code EH115 (Lonza, cat #VVPA-1002). Immediately after electroporation, pre-warmed media was added to each electroporation well, and 96-well plate was placed at 37 degrees for 20 minutes. Cells were then transferred to culture vessels in X-Vivo media containing 50U/mL IL-2 at 1e6 cells /mL in appropriate tissue culture vessels. Two days later, 1.5ug/mL Puromycin was added in culture media for selection. Cells were expanded every two days, adding fresh media with IL-2 at 50U/mL. Cells were maintained at a cell density of 1e6 cells /mL. On the final day (Day 13) of the experiment, cells from each of the nine donors were counted using Vi-CELL XR and pooled at equal numbers to obtain a final 180,000 cells in 60 uL of PBS. The pooled cells were then processed by UCSF Institute for Human Genetics (IHG) Genomics Core using 16 wells of 10X Chromium Single Cell v2 (PN-120237), as per manufacturer's protocol, with each well being separately index.