Hybrid protocol for Nuclei Isolation and 10X Genomics Single Cell 5' Gene Expression for Human Ovary Explants
Nicolas Martin
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.
Steps
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.
http://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.
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.
http://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.
The protocol CG000474 Rev B was used for Automated Gene expression dual index library construction from cDNA inputs.
http://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/library-prep/automated-gene-expression-library-construction
