Question: Are protocol modifications recommended when using the Chromium Nuclei Isolation Kit with poor quality or RNase-rich tissues?
Answer: Adding RNase Inhibitor to the Lysis Buffer may improve gene expression data quality when using poor quality or RNase-rich tissues with the Chromium Nuclei Isolation Kit. This supported protocol modification is described in further detail below.
While many tissue types have been run successfully using the Chromium Nuclei Isolation Kit without this protocol modification (see tested tissues list), through recent in-house testing we found that this protocol modification may improve gene expression data quality when working with RNase-rich tissues or lower quality samples. Improvements to data quality may vary based on the starting sample type and quality.
Additional suggestions regarding RNase-rich tissues can be found here: Can I use RNase-rich samples for single cell gene expression or Multiome assays?
Protocol modification:
Add 10x Genomics RNase Inhibitor 40X (Kit PN-1000887, Tube PN-2001488) to a final concentration of 1X in Lysis Buffer. Exclude an equal volume of Lysis Reagent from the master mix. These changes are reflected in the “Lysis Buffer (with RNase Inhibitor)” formulation table below. No other protocol modifications are required when proceeding with nuclei isolation following the Chromium Nuclei Isolation Kit Sample Prep User Guide (CG000505).
It is not recommended to add RNase inhibitors other than 10x Genomics RNase Inhibitor 40X to the Lysis Buffer. The standalone 10x Genomics RNase Inhibitor 40x (Kit PN: 1000887, Tube PN: 2001488) is not interchangeable with the RNase Inhibitor (PN: 2000565) included in the Chromium Nuclei Isolation Kit.
This protocol modification is not recommended for use with ATAC v2, but is feasible with all other assays compatible with Chromium Nuclei Isolation Kit. See this Q&A article for a list of assays compatible with the Chromium Nuclei Isolation Kit.
3’ Gene Expression Data Highlight:
Nuclei from frozen mouse liver were isolated using the Chromium Nuclei Isolation Kit, with Lysis Buffer either with or without 10x Genomics RNase Inhibitor. GEM-X Universal 3’ v4 Gene Expression libraries were generated from each nuclei suspension.
Figure 1: cDNA Bioanalyzer traces from nuclei lysed without (left) or with (right) 10x Genomics RNase Inhibitor 40X. Evidence of RNA degradation is observed when using Lysis Buffer lacking RNase Inhibitor, and addition of RNase Inhibitor increases the integrity of the RNA.
Figure 2: Gene complexity curves. Approximately 60% more genes (at 20,000 reads per cell) with 10x Genomics RNase Inhibitor added to Lysis Buffer. The magnitude of the impact will vary depending on the tissue type and quality.
Multiome Data Highlight:
Nuclei from frozen mouse small intestine were isolated using the Chromium Nuclei Isolation Kit, with Lysis Buffer either with or without 10x Genomics RNase Inhibitor. Multiome ATAC + Gene Expression libraries were generated from each nuclei suspension.
Figure 3: cDNA Bioanalyzer traces from nuclei lysed without (left) or with (right) 10x Genomics RNase Inhibitor 40X. Significant evidence of RNA degradation is observed when using Lysis Buffer lacking RNase Inhibitor, and addition of RNase Inhibitor increases the integrity of the RNA.
Figure 4: Gene complexity curves. Approximately 700% more genes (at 20,000 reads per cell) with 10x Genomics RNase Inhibitor added to Lysis Buffer. The magnitude of the impact will vary depending on the tissue type and quality.
Products: Epi Multiome ATAC + Gene Expression, Universal 3' Gene Expression, Universal 5' Gene Expression, Flex Gene Expression