Mar 15, 2024

Public workspaceIsolation of NeuN+ cells from brain tissue (for CUT and RUN)

DOI
dx.doi.org/10.17504/protocols.io.4r3l27pejg1y/v1
  • 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.
Raquel Garza
Open access
DOI: dx.doi.org/10.17504/protocols.io.4r3l27pejg1y/v1
Protocol Citation: anita.adami 2024. Isolation of NeuN+ cells from brain tissue (for CUT and RUN). protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27pejg1y/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: March 03, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 78067
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 describes the steps to isolate NeuN+ cells from brain tissue in preparation for CUT and RUN
Isolation of NeuN+ cells
Isolation of NeuN+ cells
45m
Nuclei were isolated from frozen tissue as described here.
Before FACSing, nuclei were incubated with Recombinant Alexa Fluor® 488 Anti-NeuN antibody [EPR12763] - Neuronal Marker (Abcam, Cat# ab190195, RRID:AB_2716282) at a concentration of 1:500 for Duration00:30:00 TemperatureOn ice as according to Spalding, K.L. et al. (2013).

30m
The nuclei were run through the FACS at Temperature4 °C with a low flow rate using a 100 mm nozzle and 300.000 nuclei Alexa Fluor – 488 positive nuclei were sorted.

The sorted nuclei were pelleted at 1,300 x g for Duration00:15:00 and resuspended in 1 mL of ice-cold nuclear wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, 1x cOmplete protease inhibitors, 0.1% BSA) and 10 µL per antibody treatment of ConA-coated magnetic beads (Epicypher) added with gentle vortexing (Pipette tips for transferring nuclei were pre-coated with 1% BSA), to perform CUT&RUN.

15m