May 14, 2025
JAX_DPC: scRNA sequencing using 10X Chromium Fixed RNA Profiling
- anahita.amiri 1,
- Jessica Grassmann1,
- grace.frohock Frohock1,
- Allison McNeilly1,
- Elise Courtois1
- 1The Jackson Lab
- MorPhiC Consortium
Protocol Citation: anahita.amiri , Jessica Grassmann, grace.frohock Frohock, Allison McNeilly, Elise Courtois 2025. JAX_DPC: scRNA sequencing using 10X Chromium Fixed RNA Profiling. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkrpj1v5r/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: May 13, 2025
Last Modified: May 14, 2025
Protocol Integer ID: 218201
Keywords: using 10x chromium fixed rna profiling, chromium fixed rna profiling reagent kit, 10x chromium fixed rna profiling this protocol, cell gene expression analysis of multiplexed sample, 10x genomic, cell gene expression analysis, cortical brain organoid, rna, scrna, gene, multiplexed sample
Funders Acknowledgements:
JAX MorPhiC DPC
Grant ID: 1 UM1 HG012651-01
Abstract
This protocol outlines the use of the Chromium Fixed RNA Profiling Reagent Kits (10x Genomics) for single-cell gene expression analysis of multiplexed samples derived from cortical brain organoids, across a three-day consecutive time course.
Troubleshooting
Procedure
Fixation of Cells
Dissociate differentiated iPSCs into single cells
Centrifuge sample at 350 rcf for 5 min at 4°C.
Remove the supernatant without disturbing the pellet.
Add 1 ml Fixation Buffer to the sample pellet and pipette mix 5x.
Incubate for 1 h at room temperature (20°C) or for 16-24 h at 4°C for long term storage.
Centrifuge at 850 rcf for 5 min at room temperature.
Remove the supernatant without disturbing the pellet.
Add 1 ml chilled Quenching Buffer to the sample pellet and pipette mix 5x and keep on ice.
Determine cell concentration of the fixed sample using AO/PI (acridine orange/propidium iodide) Cell Viability Kit for Luna-FL automated cell counter.
Thaw Enhancer (10x Genomics PN-2000482) for 10 min at 65°C. Vortex and centrifuge briefly. Keep warm and verify no precipitate before use.
Add 1 volume pre-warmed Enhancer to fixed sample in Quenching Buffer. Pipette mix.
Store sample at 4°C for up to 1 week.
For long term storage (up to 6 months) also add 50% glycerol for a final concentration of 10% with Quenching buffer and store at -80°C.
Chromium Fixed RNA Profiling for Multiplexed Samples
The reagents and process workflow are described below: