Mar 15, 2024
CUT and RUN
- 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. CUT and RUN. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkwb8dl5r/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: August 29, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69297
Keywords: ASAPCRN
Funders Acknowledgements:
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 protocols describe how to perform CUT&RUN on human brain tissue (frozen)
Sample extraction
Sample extraction
Flash-freeze postmortem brain tissue
Sample 50 mg - 100 mg from human brain tissue and store at -80 °C until use
CUT&RUN
CUT&RUN
Activate ConA-coat magnetic beads (Epicypher) by washing twice in bead binding buffer [20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl, 1 mM MnCl2]. Place on ice until use.
Isolate nuclei from frozen tissue after incubating with Recombinant Alexa Fluor® 488 Anti-NeuN antibody [EPR12763] - Neuronal Marker (ab190195) at a concentration of 1:500 for 30 minutes on ice.
Run nuclei through the FACS at 4 °C with low flowrate using a 100 mm nozzle and isolate 300.000 nuclei Alexa Fluor – 488 positive nuclei.
Pellet the sorted nuclei at 1,300 x g for 00:15:00 and resuspend 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).
15m
Bind nuclei to beads for 00:10:00 at RT with gentle rotation, and then split bead-bound nuclei into three equal volumes (corresponding to IgG control, H3K4me3 and H3K9me3 treatments).
10m
Remove wash buffer and resuspend nuclei in 100 µL cold nuclear antibody buffer (20 mM HEPES pH 7.5, 0.15 M NaCl, 0.5 mM Spermidine, 1x Roche complete protease inhibitors, 0.02% w/v digitonin, 0.1% BSA, 2 mM EDTA) containing primary antibody at 1:50 dilution and incubate at 4 °C Overnight with gentle shaking.
10m
Wash nuclei thoroughly with nuclear digitonin wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete protease inhibitors, 0.02% digitonin, 0.1% BSA) on the magnetic stand.
After the final wash, add pA-MNase in nuclear digitonin wash buffer and incubate with the nuclei at 4 °C for 01:00:00 . Wash nuclei twice, resuspend in 100 µL digitonin buffer, and chill to 0 °C -2 °C in a metal block sitting in wet ice.
1h
Stimulate genome cleavage by addition of 2 mM CaCl2 at 0 ºC for 30 min. Quench the reaction by additing 100 µL 2x stop buffer (0.35 M NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 50 ng/µL glycogen, 50 ng/µL RNase A, 10 fg/µL yeast spike-in DNA) and vortex.
Incubate 00:30:00 at 37 °C to release genomic fragments. Place bead-bound nuclei on the magnet stand and purify fragments from the supernatant using a NucleoSpin clean-up kit (Macherey-Bagel).
30m
Sequencing
Sequencing
Prepare Illumina sequencing libraries using the Hyperprep kit (KAPA) with unique dual-indexed adapters (KAPA).