Jan 09, 2025
CUT and RUN - Cas9 crosslinked
Forked from 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 2025. CUT and RUN - Cas9 crosslinked . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dr5rvqe/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: January 09, 2025
Last Modified: January 09, 2025
Protocol Integer ID: 117996
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 crosslink CUT&RUN on fresh cells
Sample preparation
Sample preparation
25m
25m
Detach cultured cells with Accutase (37 °C incubation for 5 minutes, or until cells completely detach) and resuspend in washing buffer (10% knockout serum [KSR] in iPSBrew).
NOTE: For optimal outcome, it is essential to use fresh cells.
Pellet cell by centrifuging them @ 400 x g for 00:05:00 .
5m
Count cells and harvest 500 000 cells per condition (and antibody).
EXAMPLE: If you are running a CRISPRi experiment (CRISPRi gRNA hiPSCs & non-targeting control hiPSCs) and you want to perform a Cas9 CUT&RUN on both conditions, you will harvest 500 000 CRISPRi and 500 000 control hiPSCs. If you are also including CUT&RUN controls (e.g. IgG control), you will need 500 000 per condition, per antibody- in this case: 500 000+500 000 CRISPRi hiPSCs & 500 000+500 000 control hiPSCs.
CUT&RUN (crosslinked)
CUT&RUN (crosslinked)
2h 23m
2h 23m
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.
Centrifuge cells at 600 x g for 00:03:00 and resuspend the pellet in 1 ml of 0.1% formaldehyde solution to allow light crosslinking. Incubate for 00:01:00 at Room temperature .
NOTE: Crosslinking is performed to capture more challenging targets such as Cas9, but it is not needed for the CUT&RUN control samples (e.g. IgG). To perform a regular CUT&RUN, refer to the the standard CUT and RUN protocol: dx.doi.org/10.17504/protocols.io.j8nlkwb8dl5r/v1
CAUTION: Formaldehyde is classified as a carcinogen and can cause respiratory and skin irritation upon exposure. Handle under a chemical hood and with the relevant protection equipment.
4m
Add 125 mM of glycine and centrifuge again at 600 x g for 00:03:00 .
3m
Remove supernatant, and resuspend cells in800 µL of XL-Wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 1 Roche Complete Protease Inhibitor EDTA-free tablet; 1% Triton X-100; 0.05% SDS - all diluted in dH2O).
Centrifuge cells at 1000 x g for00:03:00 and resuspend again in XL-Wash Buffer. Repeat two more centrifugations at 1200 x g (3 minutes each).
3m
Bind nuclei to beads (10 µL ) for 00:10:00 at RT with gentle rotation, and then split bead-bound nuclei into three equal volumes (here, corresponding to IgG control and Cas9 antibody treatment).
10m
Remove wash buffer and resuspend nuclei in 100 µL cold XL-Antibody Buffer (XL-Wash Buffer; 0.05% digitonin; 2 mM EDTA; 1:50 anti-Cas9 antibody).
CAUTION: Digitonin is toxic. Gloves should be worn when preparing and working with digitonin.
Incubate Overnight at 4 °C on gentle shake.
3m
On the following day, remove supernatant by placing the sample(s) on a magnetic stand, and resuspend/wash the beads twice in XL-Digitonin Buffer (XL-Wash Buffer + 0.05% digitonin).
After the final wash, add pA-MNase (final concentration 700 ng/ml) in XL-Digitonin Buffer and incubate with the cells at 4 °C for 01:00:00 . Wash nuclei twice, resuspend in 100 µL XL-Digitonin Buffer, and chill to 0 °C -2 °C in a metal block sitting on wet ice.
1h
Stimulate genome cleavage by addition of 2 mM CaCl2 at 0 ºC for 00:30:00 . Quench the reaction by adding 100 µL 2x Stop Buffer (0.35 M NaCl; 20 mM EDTA; 4 mM EGTA; 0.05%
digitonin; 50 ng/ml of RNAse A; 50 ng/ml of glycogen; 10 ng/ml spike in DNA; all diluted in distilled H2O) and vortex.
30m
Incubate at 37 °C for 00:30:00 to allow the release of the fragmented chromatin.
30m
Place bead-bound nuclei on the magnet stand and collect the supernatant in a new 1.5 ml Eppendorf tube.
Add 0.09% SDS and 0.22 mg/ml of Proteinase K to reverse the crosslinking.
Isolate the DNA using NucleuSping PCR Cleanup columns (Macherey-Nagel) and prepare libraries for sequencing.
Sequencing
Sequencing
Prepare Illumina sequencing libraries using the Hyperprep kit (KAPA) with unique dual-indexed adapters (KAPA).