Isolation of Nuclei for Single Cell RNA Sequencing

Demonstrated Protocol, Last Modified on March 31, 2021, Permalink

CG000124_Demonstrated_Protocol_Nuclei_isolation_RevE.pdf

These Demonstrated Protocols describe best practices and general protocols for cell lysis, washing, debris removal, counting, and concentrating nuclei from both single cell suspensions and neural tissue in preparation for use in 10x Genomics® Single Cell Protocols. Minimizing the presence of nuclear aggregates, dead cells, cellular debris, cytoplasmic nucleic acids, and potential inhibitors of reverse transcription is critical to obtaining high quality data.

The Protocols described here are expected to be compatible with many, but not all, cell or tissue types. Additional optimization may be required for the preparation of cell or tissue types that are particularly sensitive to suspension composition or handling techniques. Preparation of single cells or isolation of nuclei direct from solid tissues or cryopreserved samples may also require additional optimization during dissociation and/or cell handling not covered here. For additional information on preparation of specific sample types, consult 10x Genomics Demonstrated Protocols available on the 10x Genomics Support site support.10xgenomics.com.

This protocol is only compatible with the Chromium Single Cell Gene Expression reagent kits. Do not use with Multiome ATAC + Gene Expression or Single Cell ATAC assays.
This demonstrated protocol is optimized for counting nuclei in the range of 700-1200 nuclei/µl. If using the Single Cell 3' LT v3.1 (low throughput) application, ensure cells are counted as indicated in this protocol and then diluted to the LT specific optimal loading concentration of 100-600 nuclei/µl using the Cell Dilution Overview in the LT User Guide.