Mar 26, 2026
Isolation of Olfactory Neurons by FACS for 10x Genomics scRNA-seq
- Daniel Barnes1
- 1Department of Neuroscience, University of Pennsylvania
Protocol Citation: Daniel Barnes 2026. Isolation of Olfactory Neurons by FACS for 10x Genomics scRNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vze1k5vx1/v1
Manuscript citation:
Barnes DT, Crenshaw EMD, Curran MJ, Herr JB, Devereaux ES, Seligman CD, et al. (2026) Delta family protocadherins contribute to protoglomerular targeting of olfactory sensory neuron axons in the olfactory bulb. PLoS Genet 22(3): e1012090. https://doi.org/10.1371/journal.pgen.1012090
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 have used this protocol and have generated high quality 10x genomics scRNA-seq data sets from zebrafish embryos.
Created: February 05, 2026
Last Modified: March 26, 2026
Protocol Integer ID: 242698
Keywords: isolation of olfactory neuron, olfactory neuron, zebrafish embryo, 72hpf zebrafish embryo
Funders Acknowledgements:
NIH grant from NIDCD
Grant ID: 5R01DC012854 (JAR PI)
Abstract
This is a protocol used to isolate OMP:RFP labeled olfactory neurons from 36-72hpf zebrafish embryos.
Materials
Materials
Sterile water (tissue culture grade) – store at 4C.
Sterile E3 (Just E3 made with TC grade sterile water)
HBSS without Ca++ and Mg++ (Gibco)
1M HEPES
Tricaine – Store at 4C.
FACSmax cell dissociation solution (Genlantis Cat#t200100)
Ultrapure BSA (#AM2616 From Thermo, This is expensive and you go through it pretty quick but it is necessary)
RNAse free 1.5ml tubes
FACS tubes (Falcon 352054)
Optional: Nylon mesh cell strainer (BD352340) / flowmi pipette tip strainer.
Solutions
E3 with 1% pen/strep
100ml Sterile E3 + 1ml pen/strep
Dissection Solution- 67% HBSS with antibiotics
Add 67ml HBSS to 32ml sterile water with 1ml Penicillin/Streptomycin. Store at 4C.
FACS medium - HBSS+25mMHEPES+1%BSA (ultrapure BSA from Thermo)
For 1ml Add: 200 ul (50mg/ml) Ultrapure BSA + 25 ul 1M HEPES + 775 ul HBSS
10x Wash Solution
200ul 50mg/ml ultrapure BSA + 800ul HBSS
10x Run solution
50ul 50mg/ml ultrapure BSA + 500ul HBSS
Troubleshooting
Before start
This protocol requires prior approval by the users Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Day 0 - Set up Fish
1h
Set up fish containing GFP/RFP markers.
Set up WT fish for negative controls during FACS sorting.
For isolating OMP:RFP OSNs about 1200 embryos need to be dissected.
1h
Day 1 - 0hpf Mate Fish
1h
Mate fish at 8:00AM for 30 minutes. Stop mating after 30 minutes and collect embryos using fresh E3 from and new petri dishes. If you are not worried about age matching or having perfectly staged embryos you can mate for longer.
1h
Day 2 - 24hpf
1h
Bleach embryos, and then keep embryos in sterile E3(with 1% pen/strep) after bleaching and using new plates. Use prewarmed media to avoid developemental delays from colder temperatures. It is important to keep things as clean as possible. Background bacterial contamination will effect scRNAseq data.
1h
Day4-6 36-72hpf
8h
1.~7am prep for dissections
A. Stage the embryos if needed.
B. Optional - Screen out non-fluorescent fish from your crosses.
C. Use pronase to dechorionate embryos if needed.
D. Prep your injection setup
a) Buckets with ice
b) Tricane
c) 67% HBSS
d) Dissection dishes
e) Sample collection tubes
f) Dissection equipment of choice. I prefer using 23g needles, scalpels also work well for older ages
2. At 8 AM tricaine fish and put dishes on ice.
3. Transfer a small number of embryos to a 35mm tissue culture dish lid containing ice-cold 67% HBSS w Ca and Mg. Add a few drops of tricaine to keep the embryos anesthetized.
4. Use a sterile 23G needle to hold embryo down and another to dissect olfactory tissue or the whole head. Transfer dissected tissue to a sterile-RNAse-free eppendorf tube using a P200 pipette with a filter tip.
5. Collect tissue from up to 50 embryos in one eppendorf tube. Then move to the next tube. Be sure to keep the tubes on ice throughout the collection. For 72hpf and older only place 30 heads per tube max.
6. Collect tissue from ~200 embryos in 4 eppendorf tubes.
7. Dissect at least 20-50 heads from WT embryos for controls.
8. Tissue will sink to the bottom of the tube. Remove as much excess medium as possible.
9. Add 500ul of FACSMax (cold) to each of the tubes. Incubate at 28.5oC for 20minutes. Perform this step in a benchtop heatblock, water bath or similar. Don’t do this in an air incubator as the sample will not warm up quickly enough. Invert the tube every 5 minutes to mix the solution and break up the heads that have all settled.
10. Triturate by pipetting up and down with a 1ml pipette tip 10 times. This helps break up large clumps and expose more cells to FACS MAX.
11. Incubate for another 10 minutes
12. Then reduce the pipette tip hole size by attaching a P200 pipette tip to the 1ml tip. Pipette up and down SLOWLY for another 10 times. The stacked pipette tips give you the volume to pull up the whole sample with the smaller size tip for breaking up tissue. The edges between the two tips also cause some turbulence as the sample passes through.
13. The cells should now be in a single cell suspension.
14. Allow tissue that has not dissociated to settle at the bottom of the tube and either pipette these clumps further or remove them to avoid clumping issues during FACS. Cell strainers can be used but I found these significantly lower yield.
15. Combine all fluorescent cell suspensions into a single 15ml falcon tube. Transfer WT cell suspension into another 15ml falcon tube. Centrifuge both suspensions for 6 mins at 2000rpm(400g) in a clinical centrifuge.
16. Cells will be pelleted to the bottom of the tube. Remove all medium and replace with 500ul FACS medium for the sample and 100-200ul for the control.
17. Triturate again by pipetting with 1ml pipette tip for 5X and P200 tip for 5X.
18. Optional: Filter cell suspensions through a nylon mesh to remove any large aggregates. This typically reduced yield and can reduce viability due to shear forces.
19. Transfer the two cell suspensions to FACS tubes and take to FACS facility on ice.
20. Also take eppendorf tubes containing FACS media buffer (300ul) on ice. Cells will be collected directly in this buffer.
21. Perform FACS. Use the WT samples for background gating (FSC, SSC, and background fluorescence).
Sort using stringent gating to obtain OMP:RFP neurons. For my 10x experiment with OMP:RFP neurons I was very stringent with the goal of only capturing the best cells. Damaged or dying cells will generate background reads later on, they will increase sequencing costs ans possibly create problems for the 10x Chromium controller.
22. Pellet the collected cells by centrifugation at 400g for 6 minutes and then remove the FACS sorting solution.
23. Resuspend in 1ml 10x wash media. This wash is to removed HEPES which can impact 10x Genomics chemistry downstream.
24. Pellet cells by centrifugation at 400g for 3 minutes and remove the 10x wash media. Resuspend cells in 10x run Media. Choose your resuspension volume carefully. From FACS you will know roughly how many cells you have successfully sorted. The final cell concentration is important for the 10x controller to be able to efficiently pair the 10x beads with a cell. There is a chart with efficiencies in the 10x manual that will suggest volumes for cell suspensions to achieve desired cell concentrations.
8h