FFPE blocks were obtained from Avaden Biosciences (human brain, human lung, human lymph node, human skin and human cervix), Discovery Life Sciences (human colon and human breast) and BioIVT (human kidney). 25 micron FFPE curls were collected from each of the tissues, and cells were isolated following the demonstrated protocol for Sample Preparation from FFPE Tissue Sections for GEM-X Flex Gene Expression (CG000784), using the gentleMACS Octo Dissociator option.
After dissociation, samples were hybridized overnight and then 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 needed 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 using the following scheme:
- 28 cycles Read 1
- 10 cycles i7
- 10 cycles i5
- 90 cycles Read 2