Dec 13, 2024
JAX-Sen: Nuclei prep from frozen heart tissue for single-nuclei RNA-seq
- Sandra L Daigle1,
- Greggory A Perry1,
- William F Flynn2,
- Elise T Courtois2
- 1Single Cell Biology Lab, The Jackson Laboratory, USA;
- 2The Jackson Laboratory for Genomic Medicine
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Sandra L Daigle, Greggory A Perry, William F Flynn, Elise T Courtois 2024. JAX-Sen: Nuclei prep from frozen heart tissue for single-nuclei RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwy6zv8j/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: December 13, 2024
Last Modified: December 13, 2024
Protocol Integer ID: 115132
Funders Acknowledgements:
National Institute on Aging (NIA) JAX-Sen Senescence Tissue Mapping Center Grant
Grant ID: U54 AG079753
Abstract
We aim to study and characterize senescence in the C57BL/6J mouse heart. This protocol describes the nuclei prep from frozen heart tissue for single-nuclei RNA-seq.
Abstract
Abstract
We aim to study and characterize senescence in the C57BL/6J mouse heart. This protocol describes the nuclei prep from frozen heart tissue for single-nuclei RNA-seq.
Reagents and Materials
Reagents and Materials
Nuclei Isolation
- Chromium Nuclei Isolation with RNase Inhibitor Kit: 1000494 (10x Genomics)
- MACS BSA Stock Solution: 130-091-376 (Miltenyi)
- DPBS: 14190144 (Thermo Fisher Scientific)
- Eppendorf‱ DNA LoBind1.5 mL Microcentrifuge Tubes: 13-698-791 (Thomas Scientific)
- Lobind Eppendorf 2.0mL tubes: 13-698-792 (Thermo Fisher Scientific)
- BOApluriStrainer Mini 70 µm: 43-10070-50 (pluriSelect)
- BOApluriStrainer Mini 40 µm: 43-10040-50 (pluriSelect)
- Anti-Nucleus Microbeads: 130-132-997 (Miltenyi)
- LS Columns: 130-042-401 (Miltenyi)
- QuadroMACS Separator: 130-090-976 (Miltenyi)
- MultiStand: 130-042-303 (Miltenyi)
Cell Counting: LUNA FX7
- Acridine, Orange/Propidium Iodide Stain: F23001(New England BioGroup, LLC)
- Luna Slides:
- Ultra-low Fluorescent Slides: Fluorescent and Brightfield Mode (New England BioGroup, LLC)
Procedure
Procedure
Before Starting:
Pre-cool centrifuge, buffers, and consumables with sample contact ( columns, tubes and Strainers, etc.) at 4 °C.
Prepare Buffers
1 Heart tissue-tissue cut in quarters before snap freezing-each quarter per column
4 columns per 1 Heart x 2 hearts per batch (8 total columns)
Chromium Procedure for Tissue Lysis and nuclei extraction
Transfer frozen tissue (1/4 heart) to pre-chilled 1.5mL tube on ice
Add 200µL Lysis Buffer and dissociate the tissue using plastic pestle, leave on ice. Continue with the rest of the samples in the batch
Add additional 300µL Lysis Buffer. Pipette mix 10x, if the tip clogs do additional pestling
Incubate on ice for 15min
Pipette lysate into pre-chilled 10x columns (2mL tube)
Centrifuge (use fixed rotor) 1600 rcf 20 sec 4C
Discard column, mix well and Spin again 500 rcf 3 min 4C (swing bucket)
Remove supernatant - leave behind ~200µL if pellet in not visible
Debris Removal Steps
Resuspend pellet in 500µL Debris Removal Buffer, gently mix 15x
Spin 700 rcf 10 min 4C (swing bucket)
Remove supernatant - leave behind ~200µL if pellet in not visible
Resuspend pellet in 1mL Wash Buffer (WB), gently mix 15x
Spin 500 rcf 5 min 4C (swing bucket)
Remove supernatant - leave behind ~200µL if pellet in not visible
Resuspend pellet in 1mL Wash Buffer (WB), gently mix 15x
Spin 500 rcf 5 min 4C (swing bucket)
Resuspend pellet in 40-60µL (NRB), gently mix 15x combine nuclei from all 4 tissues from 1 heart
Optional: use 40µm if samples are clumpy
Count