Apr 02, 2024
Protocol for Nuclei Isolation and Automated 10X Genomics Single Cell 5' Gene Expression for Human Ovary Explants
- Nicolas Martin1
- 1Buck Institute for research on Aging
Protocol Citation: Nicolas Martin 2024. Protocol for Nuclei Isolation and Automated 10X Genomics Single Cell 5' Gene Expression for Human Ovary Explants. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r6x5g1b/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
Protocol status: Working
We use this protocol and it's working
Created: March 29, 2024
Last Modified: April 02, 2024
Protocol Integer ID: 97505
Keywords: gene expression for human ovary explant, automated 10x genomics single cell, 10x genomics automated single cell, gene expression protocol, 10x genomic, gem generation to final gene expression library, protocol for nuclei isolation, gene expression, final gene expression library, human ovary explant, fresh frozen human ovary explant, nuclei isolation, using nuclei suspension, nuclei suspension, cell, nuclei, 10x genomics single cell, frozen human ovary explant
Disclaimer
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).
Abstract
This is the 10X Genomics Automated Single Cell 5' Gene Expression protocol using nuclei suspension isolated from fresh frozen human ovary explants. The Chromium Connect (PN-1000171) was used from Gem generation to final gene expression libraries.
Guidelines
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).
Materials
Refers to the various protocol documents for a complete list of the materials required.
Nuclei Isolation Protocol for Human Ovary Explants
Chapter 1—Single Cell Gene Expression & Chromium Fixed RNA Profiling of the protocol CG000505 REV A was used to isolate nuclei from frozen human ovary explants with the following modifications: 1) a cordless motor pestle (VWR, Catalog number 47747-370) was used for Step f, Page 30 and 2) the samples were incubated for 15 min on ice for Step h, Page 30.
Chromium Next GEM Automated Single Cell 5' Reagent Kits v2
The Protocol CG000384 Rev D was used to generate gene expression libraries from nuclei suspension inputs.
Protocol references
The following protocols from 10X Genomics were used for the different steps:
Nuclei Isolation: CG000505 REV A
Chromium Next GEM Automated Single Cell 5' Reagent Kits v2: CG000384 Rev D