Version 1.0
A PDF version of this protocol is available for download here.
Single Nuclei Preparation for Mouse Eye
This protocol was used in
5 Million Mouse Single-Cell Atlas from 7 Tissues
Materials
| Material | Supplier | Part Number |
| Sucrose | Sigma-Aldrich® | S0389 |
| KCl | Invitrogen® | AM9640G |
| MgCl2 | Invitrogen | AM9530g |
| Tris buffer, pH 8 | Invitrogen | AM9856 |
| Nuclease-Free Water | Sigma-Aldrich | W4502 |
| DTT | Invitrogen | P2325 |
| RNase Inhibitor | QIAGEN® | Y9240 |
| SUPERaseᐧIn RNase Inhibitor | Invitrogen | AM2696 |
| Triton X-100 | Sigma-Aldrich | T8787 |
| DNA low binding, DNase/RNase free, filtered pipette tips 20 μL, 200 μL, 1,000 μL | Varies | Varies |
| Falcon® High Clarity PP Centrifuge Tubes, 15 mL | Corning® | 352097 |
| 5.0mL LoBind® Tubes | Eppendorf® | 0030108302 |
| Cell Strainer (40 μm) | Corning | 431750 |
| Trypan Blue | Varies | Varies |
| RNaseZAP® RNase Decontamination Solution | Thermo Fisher Scientific® | AM9780 |
| Ethyl alcohol, Pure | Sigma-Aldrich | 459844 |
| Optional: Fluorescent stain (e.g., Hoechst, DAPI, or AOPI) | Varies | Varies |
| Dounce Tissue Grinder, 1mL | WHEATON® | 357538 |
| Single channel pipettes: P20, P200, P1000 | Varies | Varies |
| RNAse-free forceps | Varies | Varies |
| Hemocytometer | Sigma-Aldrich | Z359629 |
Preparation
1. Clean the bench top and dissection area with 70% ethanol, followed by RNaseZAP to remove RNases.
2. Prepare the following solutions, and add reagents in the order provided in the table.
3. Clean the Dounce homogenizer with RNaseZAP followed by 70% ethanol then rinse well with dH2O and let dry.
NIM1 buffer
| Reagents | Stock Concentration | Final Concentration | Volume for 15 mL |
| Sucrose | 1.5 M | 250 mM | 2,500 μL |
| KCl | 2 M | 25 mM | 187.5 μL |
| MgCl2 | 1 M | 5 mM | 75 μL |
| Tris buffer, pH 8 | 1 M | 10 mM | 150 μL |
| Water | - | - | 12,087.5 μL |
Homogenization buffer
| Reagents | Stock Concentration | Final Concentration | Volume for 5 mL |
| NIM1 Buffer | 1.5 M | 4,845 μL | |
| DTT | 1 mM | 1 µM | 5 μL |
| RNase Inhibitor | 40 U/µL | 0.4U/µL | 50 μL |
| SUPERaseᐧIn RNase Inhibitor | 20 U/µL | 0.2U/µL | 50 μL |
| Triton X-100 | 10% | - | 50 μL |
Note: If there is a potential that your sample was exposed to any external sources of RNases during tissue harvest or if your tissue is known to have elevated RNase activity, you can optionally add in additional RNase inhibitors of your choosing when preparing the homogenization buffer.
Nuclei Extraction
1. Gather a dounce and keep at 4°C until use.
2. Make the NIM1 buffer and keep on ice until use.
3. Make the homogenization buffer and keep on ice until use.
4. Dounce. The mouse eyeball can be placed directly into the dounce to minimize cell loss.
a. Add tissue sample to the dounce and resuspend in 700 µL of homogenization buffer.
b. Perform 5 strokes of loose pestle.
c. Perform up to 10 - 15 strokes of tight pestle. Add additional strokes of the tight pestle if you still see large pieces.
d. Add additional homogenization buffer up to 1.4 mL.
5. Filter homogenates with a 40 µm strainer into a 5mL or 15 mL tube.
6. Count the nuclei using a preferred accurate method.
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 20× and 40x objective on an epifluorescence microscope. A best practices resource for hemocytometer use, see: https://www.hemocytometer.org/
7. Move up to the maximum number of nuclei* allowed by the EvercodeTM Fixation kit into the nuclei fixation workflow.
*Up to 1 million nuclei per reaction for Evercode Nuclei Fixation v3 and up to 100,000 nuclei for Evercode Low Input Nuclei Fixation v3.
Notice
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