Peripheral blood mononuclear cells (PBMCs) were obtained by 10x Genomics from AllCells. 10,000,000 cells were thawed and fixed for 1 hour at room temperature, following the demonstrated protocol for Fixation of Cells & Nuclei for GEM-X Flex Gene Expression (CG000782).
Cells were then sub-pooled across 16 different Probe Barcodes (BC001-BC016) by dividing them into 300,000-cell aliquots and hybridizing overnight, following the GEM-X Flex Gene Expression Reagent Kit for Multiplex Samples (CG000787). Hybridized samples were washed using the Pool Wash Workflow (Option A) as described in the user guide, with one modification: after the final resuspension, the sample volume was measured using a P1000 pipette, centrifuged, and the supernatant was reduced to leave 150 µL. The samples were then resuspended, counted, and loaded into a Chromium GEM-X Chip. Note: A final cell concentration of approximately 14,000 cells/µL or higher is required to target 320,000 cells.
The Gene Expression library for GEM-X Flex was generated using the GEM-X Flex Gene Expression Reagent Kit for Multiplex samples (CG000787). The Gene expression library was sequenced on an Illumina NovaSeq 6000 to a read depth of approximately 20,000 mean reads per cell.
Paired-end, dual indexing libraries were sequenced with the following scheme:
- 28 cycles Read 1
- 10 cycles i7
- 10 cycles i5
- 90 cycles Read 2