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:
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