Protocol Citation: William F Flynn, Elise Courtois, Greggory A Perry, Sandra L Daigle, Paul Gabriel, Diane Luo, Jessica Grassmann 2023. JAX - Nuclei Isolation for 10x Genomics A. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1n9npgr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
The purpose of this protocol is to produce single nuclei from frozen human tissues for downstream assaying with the 10x Genomics Multiome assay.
This protocol has been demonstrated using Human placenta tissue as well as human Glioblastoma.
This protocol is modified from Sigma Aldrich Nuclei Isolation Kit: EZ Prep protocol and 10x Chromium Demonstrated protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365).
Guidelines
Assess nuclear membrane integrity (refer to 10x Genomics CG000365), using Brightfield or Trypan blue
Assess for debris, if large amount of debris present, filter with smaller pore size to decrease debris.
Use either Trypan blue and Countess (for clean average size nuclei preps) or AO/PI and Nexcellom K2 counter (if nuclei prep has debris)
If RNA content is of concern Imaging nuclei can be done with fluorescence, and Syto RNA stain