Single Nuclei Preparation for Mouse Eye developed by Parse Biosciences
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
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.
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PDF version of protocol: