Mar 15, 2024
Nuclear Extraction from Tissue for FACS
- 1Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, BMC A11, Lund University, 221 84 Lund, Sweden.
Protocol Citation: anita.adami 2024. Nuclear Extraction from Tissue for FACS. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8j678g2w/v1
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: Working
We use this protocol and it's working
Created: February 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 76528
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000520
Swedish Research Council
Grant ID: 2018-02694
Swedish Brain Foundation
Grant ID: FO2019-0098
Cancerfonden
Grant ID: 190326
Barncancerfonden
Grant ID: PR2017-0053
NIHR Cambridge Biomedical Research Centre
Grant ID: NIHR203312
Swedish Society for Medical Research
Grant ID: S19-0100
National Institutes of Health
Grant ID: HG002385
Swedish Research Council
Grant ID: 2021-03494
Swedish Research Council
Grant ID: 2020-01660
Abstract
This protocol details the process for pre-tissue preparation and tissue preparation for nuclear extraction for FACS.
Before start
Keep everything On ice.
Pre-Tissue Preparation
Pre-Tissue Preparation
Coat all tubes (centrifugation, sample and collection tubes) in coating medium Overnight (1 % (v/v) BSA , filtered).
Cool ultracentrifuge to 4 °C prior to centrifugation
Pre-weigh the DTT needed.
Keep douncers On ice .
Tissue Preparation
Tissue Preparation
10m
Put 2.8 mL sucrose solution in centrifuge tube.
Mix 6 mL sucrose + 2 mL lysis solution in 15 ml falcon tube.
If the tissue is large (>2 mm3), chop the tissue with a razor.
Collect chopped tissue with 0.5 mL lysis buffer and transfer to douncer. Repeat with another 0.5 mL lysis buffer to get all tissue.
Dounce: 5-7x loose, 5-7x tight.
Pour douncer content into falcon tube with sucrose/lysis buffer mixture and flip upside down a few times. Pour 1 mL of tube content back and forth from douncer to collect remaining nuclei.
Carefully layer the 8.5 mL mix of lysis/sucrose/nuclei to the centrifuge tube, on top of sucrose.
Balance the tubes (≤5 mg difference).
Place tubes in ultracentrifuge, spin at 15500 rpm , 02:15:00, (rotor dependent, use 30.000xg) .
Carefully aspirate off supernatant without disturbing the pellet.
Note
Due to the viscosity, try to aspirate the inside of the tube to get rid of all supernatant.
Soften pellet for 00:10:00 in 50 µL medium A .
10m
Add 100 µL dilution buffer (containing a nuclei marker, such as Draq7) and filter using 70um pipette tip filters into coated tubes.
FACS sort, low flowrate, largest nozzle, cooling system on. The sucrose will cause problems at FACS. Avoid this by adding dilution buffer (with your nuclei marker), 100 µL at the time (usually need 300 µL -400 µL for dilution to avoid problems – add that even before starting to sort).
For single nuclei sequence using 10x: Pre-coat the pipette tips needed to load the 10x machine according to step 1.
For RNA extraction: Pellet nuclei at 1300 x g , 4°C, 00:15:00 . Remove supernatant and snap freeze for later RNA extraction (or resuspend in 350 µL RLT including beta-mercapto ethanol directly. Use RNeasy micro columns. For very small volumes, resuspend in RLT without pelleting).