GSM7653180: ESI017 WT SP8 (hDLL4 Stroma), replicate 1, VDJ scRNA-seq; Homo sapiens; RNA-Seq
Isolation of ATO-derived SP8: ATOs were harvested by adding MACS buffer (PBS, 0.5% bovine serum albumin, 2mM EDTA) to each filter, briefly disaggregating the ATO by pipetting with a 1mL “P1000” pipette, and then passed through a 50µm nylon strainer; SP8 T cells were harvested after 6 Weeks of T cell differentiation, and DP precursors were harvested after 3 Weeks. For bulk-scale collection of ATOs, aggregates were harvested in a similar fashion; however, up to 150 aggregates were collected in a well filled with MACS buffer and then transferred onto a 50µm nylon filter, where aggregates were physically dissociated on top of the filter using the back end of a sterile 1mL syringe. After dissociation, the filter was washed with MACS buffer, and cell mixtures were centrifuged at 300 x g for 5m at 4C in a swinging bucket centrifuge. Single cell RNA library preparation and sequencing: ATO-derived T cells were isolated as described above and then FACS-sorted for ATO-derived SP8 T cells from WT (mCD29-CD45+CD8a+CD4-) and 1G4 TCR-transduced (mCD29-mTagBFP2+CD45+CD8a+CD4-) PSCs ATOs into PBS + 0.04% bovine serum albumin. For ESI017 WT PSCs differentiated in MS5-hDLL4 stroma ATOs, SP8 and CD45+ fractions were both collected. Cells were counted and resuspended at a concentration of ~1000 cells/µL and provided to the Technology Center for Genomics and Bioinformatics (TCGB) core for unique molecular identifier (UMI) tagging and generation of gene expression (GEX) and human TCR repertoire (VDJ) libraries using the 10X Chromium Next GEM Single Cell V(D)J Reagent Kit v1.1 (10X Genomics, Pleasanton, CA). Similarly, peripheral blood (PB) SP8s were FACS-isolated (CD45+CD8A+CD8B+CD4-TCRab+CD3+) from PB mononuclear cells obtained from healthy donors, and provided to the TCGB core for UMI tagging and library generation. Fully constructed libraries for all samples were run in one S4 flowcell on the Illumina Novaseq Platform. 5' Gene Expression (GEX), VDJ, and Feature Barcoding libraries were constructed according to the manufacturer's instructions (Chromium Next GEM Single Cell 5' Reagent Kits v2, 10X Genomics).