Question: How can I isolate nuclei for 3' Gene Expression profiling?
Answer: In general, using whole-cell preps for the 3' Single Cell Gene Expression assay is preferred over nuclei. However, nuclei isolation is appropriate when:
a) The tissue has been frozen and dissociation is not possible. The freezing process may have compromised the cell membrane. However, the nuclear membrane is usually still intact
b) The maximum cell size we have tested in-house is 30 um. Larger cells may clog the microfluidic channels of the 10x Genomics chips. See: What is the range of compatible cell sizes?
Protocols for nuclei isolation from fresh cells and tissues:
A Demonstrated Protocol for the isolation of nuclei from single-cell suspensions and fresh neural tissue is available on the Support website. This protocol describes the best practices and general protocols for cell lysis, washing, debris removal, counting, and concentrating nuclei in preparation for use in the 10x 3' Gene Expression protocol.
Note: this demonstrated protocol is not to be used with the ATAC assay or the Multiome ATAC+Gene Expression assay.
Protocols for nuclei isolation from frozen tissues:
10x Genomics Recommends using the Chromium Nuclei Isolation kit for isolating nuclei from frozen tissues samples. The Chromium Nuclei Isolation Kit is an all-in-one sample preparation kit for generating high-quality nuclei suspensions from frozen tissue for use with Single Cell Gene Expression assays. The Chromium Nuclei Isolation kit has broad applicability across tissue types which reduces requirements for sample preparation optimization or the need for tissue-specific protocols. The short nuclei isolation protocol with this kit reduces sample degradation and results in improved data quality. The user guide can be found here: Chromium Nuclei Isolation Kit Sample Prep User Guide
If not using the Chromium Nuclei Isolation Kit, there is a customer-developed protocol for frozen tissues is available on the 10x Community site. The 'Frankenstein protocol for nuclei isolation from fresh and frozen tissue' describes nuclei isolation and sorting.
Note: Customer-Developed protocols are provided for general information only and are NOT directly supported, endorsed, or certified by 10x Genomics.
For general guidelines and tips on isolating nuclei from frozen tissues, please see: How do you isolate nuclei from snap-frozen tissue for 3’ gene expression profiling?
For best practices for working with nuclei prep, please see: What are the best practices for working with nuclei samples for 3' single-cell gene expression?
Products: Single Cell Gene Expression