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Version 1.0
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Single Nuclei Isolation from Embryonic Chicken Eye
Developed by Jared Tangeman from the Del Rio-Tsonis Lab at Miami University
About the Del Rio-Tsonis Lab
The Del Rio-Tsonis Lab explores eye regeneration, focusing on pigment epithelial cells reprogramming into lens and retina cells. Using models such as chicks and salamanders, the team investigates key genes, signals, and epigenetic regulators driving this process. Their ultimate goal is to apply these insights to retinal degenerative diseases.
This protocol was used in
Tangeman, J.A. et al. Integrated single-cell multiomics uncovers foundational regulatory mechanisms of lens development and pathology. Development 1 January 2024; 151 (1): dev202249. doi: https://doi.org/10.1242/dev.202249
Materials
- Materials | Supplier | Part Number | Notes
- Hoechst-33342 | Thermo Fisher Scientific® | H3570
- 20 µm strainer | pluriSelect® | 43-10020-40
- Tris-HCl | Millipore Sigma® | T2194 | or equivalent
- NaCl | Millipore Sigma | 59222C | or equivalent
- MgCl2 | Millipore Sigma | M1028 | or equivalent
- Surfact-Amps- NP-40 | Thermo Fisher Scientific | 28324
- Nuclease-free water | Varies | Varies
- 1X PBS | Gibco® | 10-010-031 | Or equivalent
- 10% BSA (Fraction V) | Varies | Varies
- RNase Inhibitor | Promega® | 12615
- Centrifuge with swinging bucket rotor | Varies | Varies | Compatible with 15 mL or 5 mL centrifuge tubes and capable of reaching 4°C.
- Fire-polished silanized pasteur pipette or Regular-bore pipette tips | Varies | Varies
- Hemocytometer | Varies | Varies
- Epifluorescence microscope | Varies | Varies | 20× and 40× objectives
- DAPI or AOPI | Varies | Varies | Optional nuclear stains
Preparation
1. Prepare the following solutions and add the reagents in the order provided in the table. Keep them on ice.
2. Clean the bench top and dissection area with 70% ethanol, followed by RNaseZAP to remove RNases.
Cell Lysis Buffer
- Reagents | Stock Concentration | Final Concentration | Total Volumes for 10 mL
- Tris-HCL | 1 M | 10 mM | 100 μL
- NaCl | 1 M | 10 mM | 100 μL
- MgCl2 | 1 M | 3 mM | 30 μL
- Surfact-Amps- NP-40 | 10% | 0.01% | 10 μL
- RNase Inhibitor | 40 U/μL | 0.2 U/μL | 50 μL
- Nuclease-free water | 9.71 mL
Nuclei Wash Buffer
- Reagents | Stock Concentration | Final Concentration | Volumes for 10 mL
- PBS | 10X | 1X | 1 mL
- 10% BSA, Fraction V | 10% | 2% | 2 mL
- RNase Inhibitor | 40 U/μL | 0.2 U/μL | 50 μL
- Nuclease-free water | 6.95 mL
Procedure
Critical: All steps must be performed on ice or at 4°C.
1. Add ice-cold lysis buffer to the dissected tissue.
2. Triturate gently to lyse open the cells by pipetting up and down with either a fire-polished silanized Pasteur pipette or a regular-bore pipette tip to break the tissue into small pieces.
3. Centrifuge lysate at 500 x g for 5 min at 4°C to pellet the nuclei.
4. Use a pipette to carefully remove the supernatant.
5. Resuspend the nuclei pellet in 1 mL of ice-cold Nuclei Wash buffer.
6. Repeat wash 3x, centrifuging each time at 500 x g for 5 min at 4°C.
7. Pass nuclei through a 20 µm cell strainer to filter debris and cell aggregates.
8. Repeat the straining step again, for a total of 2 strains.
9. Count the nuclei.
Note: In our experience, nuclei are much stickier than whole cells. For researchers making the single-nuclei suspension for the first time, we suggest confirming that the sample contains mostly single nuclei. To do this, take 10 μL of the sample and stain it with Hoechst, DAPI, or AOPI for more than 5 min, place it on a hemocytometer, and visualize it under a 20X and 40X objective on an epifluorescence microscope. A best practices resource for hemocytometer use, see: https://www.hemocytometer.org/
10. Proceed to the appropriate Parse Biosciences Nuclei Fixation Kit User Guide (either standard Nuclei Fixation, Low-Input Nuclei Fixation).
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