Human bone marrow aspirates from a healthy male aged 60-65 were obtained by 10x Genomics from StemExpress. Mononuclear cells were isolated, and cells were labeled with TotalSeq™-C Human Universal Cocktail, v1.0 (BioLegend) and viable cells were identified by 7AAD staining and sorted via FACS.
Gene Expression, Cell Surface Protein/Immune Receptor Mapping, BCR amplified, and TCR amplified libraries were generated from ~16,000 cells (8,288 cells recovered) as described in the Chromium Single Cell 5' Reagent Kits User Guide (v2 Chemistry Dual Index) with Feature Barcode technology for Cell Surface Protein and Immune Receptor Mapping (CG000330 Rev C) using the Chromium X. Libraries were sequenced on an Illumina NovaSeq 6000 to a read depth of 41,000 mean reads per cell for Gene Expression, 12,000 mean reads per cell for TCR amplified, 90,000 mean reads per cell for BCR amplified, and 5,000 mean reads per cell for Cell Surface Protein/Immune Receptor Mapping libraries.
Paired-end, dual indexing:
- Read 1: 26 cycles (16 bp barcode, 10 bp UMI)
- i5 index: 10 cycles (sample index)
- i7 index: 10 cycles (sample index)
- Read 2: 90 cycles (transcript)
Analysis parameters used: expect-cells=10000