Aggregate of Human Kidney Nuclei Comparing Next GEM Fixed RNA Profiling vs GEM-X Flex Gene Expression at High and Low Cell Input

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 divided into three samples: two samples containing approximately 2 million nuclei each and one low-input sample containing 25,000 nuclei.

The samples were fixed for 1 hour at room temperature. One of the 2 million nuclei samples was fixed using the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling protocol (CG000478), while the remaining samples were fixed using the Fixation of Cells & Nuclei for GEM-X Flex Gene Expression protocol (CG000782).

Following fixation, probe hybridization was performed according to the respective user guides. For the samples with 2 million nuclei, 300,000 nuclei aliquots were used for hybridization, while the entire 25,000 nuclei sample was used for hybridization. After hybridization, the samples were washed and filtered according to the respective protocols, with a modification for the low-input samples: after washing, the sample volume was measured using a P1000 pipette, centrifuged, and the supernatant was reduced to leave 50 µL. The samples were then resuspended, counted, and loaded into Chromium Next GEM and GEM-X Chips. For the 300,000 nuclei samples, 4,000 nuclei were targeted, while the maximum sample volume (34.7 µL) was used for loading the low-input sample.

Fixed RNA Gene Expression libraries for Flex were generated according to the Chromium Fixed RNA Profiling for Singleplexed Samples protocol (CG000477), and 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. The outputs of each sample were then aggregated and normalized to equal sequencing depth using the cellranger aggr pipeline, generating a single set of output files containing data from all individual samples.

The sample-level web summaries from cellranger multi runs are available for download here.

This dataset is licensed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license. 10x citation guidelines available here.