Notice: This protocol is not supported by Parse Biosciences. This Customer Developed Protocol is provided for general information only and is not directly supported, endorsed, or certified by Parse Biosciences. Parse Biosciences gives no warranties and makes no claims about the provided protocol.
Version 1.0
A PDF version of this protocol is available for download here.
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).
Notice
This document and its contents may be proprietary to Parse Biosciences, Inc. (“Parse Biosciences”) and is intended solely for use by its customers in connection with the use of the product(s) described herein and for no other purpose. The products may be used solely FOR RESEARCH PURPOSES, AND MAY NOT BE USED IN ANY DIAGNOSTIC OR THERAPEUTIC USE IN HUMANS OR ANIMALS. This document and its contents shall not be used or distributed for any other purpose and/ or otherwise communicated, disclosed or reproduced in any way whatsoever without the prior written consent of Parse Biosciences. No rights are granted under this document with respect to any of Parse Biosciences’ intellectual property rights. The license to use of any products described herein is subject to a separate written agreement between Parse Biosciences and the applicable user. Parse Biosciences shall have no liability for any direct, indirect, consequential or incidental damages arising out of any failure to use the product(s) in strict compliance with the terms herein. This document may contain references to third-party sources of information, hardware or software, products, or services, or intellectual property including but not limited to trademarks, and/ or third-party web sites (collectively “Third Party Information and Intellectual Property”). Parse Biosciences does not control, and is not responsible for, any Third Party Information or Intellectual Property. The inclusion of Third Party Information or Intellectual Property in this document does not imply endorsement by Parse Biosciences of the Third Party Information or Intellectual Property or the third party in any way. A non-exhaustive list of Parse Biosciences trademarks can be found at www.parsebiosciences.com/legal/trademarks/. Parse Biosciences products may be covered by one or more patents as listed at www.parsebiosciences.com/legal/patents/. The product(s) offer by Parse Biosciences described in this document are provided for one-time use by the purchaser and may not be re-used, refurbished or resold. In addition, such product(s) may not be altered, changed or modified by anyone other than Parse Biosciences and its authorized agents, and Parse Biosciences will not be liable for any such alterations, changes or modifications.
If you are interested in adding your protocol to our sample preparation protocols page, please reach out to us at support@parsebiosciences.com.
PDF version of protocol: