Question: Which nuclei isolation protocols are supported for use with the 3' CellPlex Kit for Cell Multiplexing?
Answer: The 3' CellPlex assay has only been validated with nuclei isolated using Demonstrated Protocol CG000124, which is our supported protocol for isolating nuclei from cells or tissues for use with the Chromium Single Cell 3' Gene Expression assay.
CellPlex assay performance cannot be guaranteed if alternative nuclei isolation protocols are followed. A high-quality nuclei suspension is essential for the 3' CellPlex assay. If nuclei are damaged or over-lysed, they may not withstand the additional handling steps associated with Cell Multiplexing Oligo (CMO) labeling and washing.
Nuclei isolated from snap-frozen tissues have not been tested for compatibility with the 3' CellPlex assay. Isolating nuclei from snap-frozen tissues can be technically challenging (see further details here), and nuclei can easily become damaged.
If you are interested in performing the 3' CellPlex assay on nuclei isolated using alternative protocols or nuclei isolated from frozen tissue, we recommend performing pilot experiments to assess nuclei quality post CMO labeling and washing. If nuclei clumping or low nuclei recovery are observed after CMO labeling, optimization of upstream nuclei isolation protocols (eg. lysis time) is recommended.
Our supported nuclei isolation protocol (CG000124) uses NP-40 as a lysis agent. Alternative detergents have not been tested for compatibility with the 3' CellPlex assay.
After lysis, it is essential to wash nuclei to remove residual detergents before proceeding with CMO labeling. Carryover of detergents into the CMO labeling step may negatively impact assay performance.
For general guidance on optimizing nuclei isolation protocols and assessing nuclei quality, see: What are the best practices for working with nuclei samples for 3' single-cell gene expression?
Notes on nuclei isolation protocols for different 10x Genomics assays
Each 10x Genomics assay has unique sample input requirements. We offer different nuclei isolation protocols that are specifically optimized for each assay type. The ATAC and Multiome nuclei isolation protocols include additional detergents during the lysis and wash steps to ensure that the nuclei are partially permeabilized to allow entry of Transposase enzymes. In contrast, permeabilization of nuclei is not needed for the Single Cell 3’ Gene Expression assay.
During in-house tests, we observed poor results when performing the 3' CellPlex assay on nuclei that had been isolated using Multiome nuclei isolation protocol CG000365. We found that these nuclei formed clumps/aggregates after the CMO labeling and wash steps, and yielded poor quality data when used in the 3' CellPlex assay. These experiments highlight the importance of using a nuclei isolation protocol that is appropriate for the assay type.
Products: Single Cell Gene Expression, CellPlex