Fresh frozen human kidney tissue was obtained from Avaden BioSciences by 10x Genomics. The tissue was sectioned into two 44 mg samples and nuclei were isolated using the Chromium Nuclei Isolation Kit (CG000505). A total of 4,180,000 nuclei were obtained, which were aliquoted into a sample containing approximately 2 million nuclei.
The sample was fixed for one hour at room temperature using the Fixation of Cells & Nuclei for GEM-X Flex Gene Expression protocol (CG000782).
Following fixation, probe hybridization was performed according to the user guide. 300,000 nuclei aliquots were used for hybridization. After hybridization, the samples were washed and filtered according to the protocol. The sample was then resuspended, counted, and loaded into a Chromium GEM-X Chip. 4,000 nuclei were targeted.
Gene Expression libraries for GEM-X Flex were generated using the GEM-X Flex Gene Expression Reagent Kit for Singleplex samples (CG000786).
Libraries were sequenced on an Illumina NovaSeq 6000, with a mean read depth of approximately 20,000 reads per cell using a paired-end, dual indexing sequencing scheme:
- 28 cycles Read 1
- 10 cycles i7
- 10 cycles i5
- 90 cycles Read 2
Libraries were analyzed using the cellranger multi pipeline. Cell type annotations were generated using 10x Genomics' automated cell annotation model: human_pca_v1_beta.