Apr 01, 2024

Public workspaceHybrid protocol for Nuclei Isolation and 10X Genomics Single Cell 5' Gene Expression for Human Ovary Explants

DOI
dx.doi.org/10.17504/protocols.io.x54v928dpl3e/v1
  • 1Buck Institute for research on Aging
Nicolas Martin
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v928dpl3e/v1
Protocol Citation: Nicolas Martin 2024. Hybrid protocol for Nuclei Isolation and 10X Genomics Single Cell 5' Gene Expression for Human Ovary Explants. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v928dpl3e/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 27, 2024
Last Modified: April 01, 2024
Protocol Integer ID: 97470
Disclaimer
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).
Abstract
This is the 10X Genomics Single Cell 5' Gene Expression hybrid protocol using nuclei suspension isolated from fresh, frozen human ovary explants. The Chromium X (PN-1000331) was used for Gem Generation, and the Chromium Connect (PN-1000171) was used for preparing Gene expression libraries from cDNA inputs.
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 material required.
Nuclei Isolation Protocol for Human Ovary Explants
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.

https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/sample-prep/chromium-nuclei-isolation-kit-sample-prep-user-guide


Chromium Next GEM Single Cell 5'Reagent Kits v2 (Dual Index). GEM Generation, Barcoding, Post GEM-RT Cleanup and cDNA Amplification.
Chromium Next GEM Single Cell 5'Reagent Kits v2 (Dual Index). GEM Generation, Barcoding, Post GEM-RT Cleanup and cDNA Amplification.
Step 1 and step 2 of protocol CG000331 REV E were used for GEM Generation, Barcoding, Post GEM-RT Cleanup, and cDNA Amplification. The Chromium X was used for Gem Generation.

https://www.10xgenomics.com/support/single-cell-immune-profiling/documentation/steps/library-prep/chromium-single-cell-5-reagent-kits-user-guide-v-2-chemistry-dual-index

Chromium Next GEMSingle Cell 5'Reagent Kits v2 (Dual Index). Gene expression library construction.
Chromium Next GEMSingle Cell 5'Reagent Kits v2 (Dual Index). Gene expression library construction.
The protocol CG000474 Rev B was used for Automated Gene expression dual index library construction from cDNA inputs.

https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/automated-gene-expression-library-construction

Protocol references
The following protocols from 10X Genomics were used for the different steps:

Nuclei Isolation: CG000505 REV A

Chromium Next GEM Single Cell 5'Reagent Kits v2 (Dual Index). GEM Generation, Barcoding, Post GEM-RT Cleanup and cDNA Amplification using manual workflow: CG000331 Rev E.

Chromium Next GEMSingle Cell 5'Reagent Kits v2 (Dual Index). Automated Gene expression library construction: CG000474 Rev B.