Sep 27, 2023
KAPP-Sen TMC: Fixation of Cells and Nuclei for Chromium Fixed RNA Profiling
- Juliana Alcoforado Diniz1,
- Dylan Baker1,
- Jessica Garofalo1,
- Paul Robson1,2,3
- 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA;
- 2Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT, USA;
- 3Institute for Systems Genomics, University of Connecticut, Farmington, CT, USA
Protocol Citation: Juliana Alcoforado Diniz, Dylan Baker, Jessica Garofalo, Paul Robson 2023. KAPP-Sen TMC: Fixation of Cells and Nuclei for Chromium Fixed RNA Profiling. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9py5zg3e/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: July 28, 2023
Last Modified: September 27, 2023
Protocol Integer ID: 85646
Keywords: SenNet KAPP-Sen TMC
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Abstract
Cells are fixated prior to scRNA-seq according to 10X Genomics protocol CG000478.
Fixation of Cells & Nuclei
Fixation of Cells & Nuclei
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.