Jan 14, 2022
Nuclear extraction from endometrial tumors for single nuclei sequencing
- 1Northwestern University
- Northwestern University
External link: https://www.nature.com/articles/s41591-020-0844-1#Sec13
Protocol Citation: gracefoley 2022. Nuclear extraction from endometrial tumors for single nuclei sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.b3vfqn3n
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: In development
We are still developing and optimizing this protocol
Created: January 14, 2022
Last Modified: January 14, 2022
Protocol Integer ID: 56967
Abstract
Modified from: Slyper, M. et al. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nat. Med. 2020 26526, 792–802 (2020).
A optimized protocol for nuclear extraction from endometrial tumors. Performed with endometrial adenocarcinoma, endometrioid type, FIGO grade 1.
Nuclear extraction optimization for single nuclei RNA-seq.pptx Materials
Buffers:
2X ST Buffer
| Stock | Final | Volume for 10 mL | ||
| NaCl | 5 M | 292 mM | 584 uL | |
| Tris-HCl (pH 7.5) | 1 M | 20 mM | 200 uL | |
| CaCl2 | 1M | 2 mM | 20 uL | |
| MgCl2 | 100 mM | 42 mM | 4.2 mL | |
| Ultrapure Water | - | - | 5 mL |
NST Buffer
| Stock | Final | Volume for 10 mL | ||
| Nonidet P40 Substitute | 10% | 200 ul | ||
| BSA | 5% | 20 ul | ||
| ST Buffer | 2X | 1X | 5 ml | |
| Nuclease Free Water | - | - | 4.75 mL |
Nuclei Resuspension Buffer
| Stock concentration | Final | Volume Needed for 50 mL | ||
| BSA solution | 5% | 1% | 10 ml | |
| RNase Inhibitor | 40 U/ul | 0.2 U/ul | 0.25 ml | |
| 1X PBS | - | - | 39.75 ml |
Place frozen tissue in a well of a 6 well plate containing 1 mL of cold NST disassociation buffer.
Ensure plate is on ice
Chop tissue using a scalpel until mostly homogenized
2m
Incubate solution on ice for 5 minutes
5m
Filter the homogenized solution through a 40 uM falcon cell strainer into a 50 mL falcon tube
Use an additional 1 mL of NST buffer to rinse the well and filter
Count nuclei
Centrifuge nuclei at 300 g for 10 minutes and aspirate supernatant completely
10m
Resuspend pellet and bring the volume up to 5 mL using ST buffer
Transfer solution to 15 mL conical tube and centrifuge for 10 minutes at 300 g at 4oC
10m
10. Resuspend pellets on ice in 1 ml of the ST buffer
11. Filter through 35 um falcon strainer
12. Count nuclei
Centrifuge nuclei at 300 g for 10 minutes and aspirate supernatant completely
10m
13. Resuspend nuclei pellet in resuspension buffer to get a concentration of 1,000 nuclei per uL
Start with .5 mL per 0.2 grams of endometrial tumor tissue
Look at nuclear membranes at 40x-60x to ensure high nuclear membrane quality with minimal blebbing