Peripheral blood mononuclear cells (PBMCs) from a CMV seronegative donor, were obtained from Astarte (donor 396, catalog #: 1001-3894AP18). Cells were stained with a panel of 12 dCODE™ Dextramer® reagents (Immudex) and 17 TotalSeq™-C antibodies (BioLegend). See details in the Application Note “Redefining Cellular Phenotyping: Comprehensive Characterization and Resolution of the Antigen-Specific T Cell Response”.
Cells were 87% viable by Trypan blue stain.
The same cDNA was used to generate gene expression and cell surface protein (vdj_v1_hs_unsorted_cmvneg_5gex_protein) libraries.
Libraries were prepared following the Single Cell V(D)J Reagent Kits User Guide (CG000186 RevA).
- ~17,400 total cells loaded
- 2,769 T cells expected based on CD3D/CD3E expressing fraction in 5’GEX data
- 3,570 T cells expected based on CD3+ fraction in antibody data (estimated by counting cells above a cut off at 50% of the log2 UMI counts) - since NK and dead cells show some CD3 binding this number is higher than predicted
- 2,502 cells detected with either single TRA or TRB or both
- 74% cells with productive V-J spanning (TRA,TRB) pair
- Sequenced on an Illumina HiSeq4000 with ~30,000 read pairs per cell
- 150bp read1 (including 16bp Chromium barcode, 10bp UMI, 13bp switch oligo and transcript), 150bp read2 (transcript), and 8bp I7 sample barcode read configuration