Jan 09, 2025

Public workspaceCUT and RUN - Fresh cells

 Forked from CUT and RUN
DOI
dx.doi.org/10.17504/protocols.io.36wgqdb83vk5/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.
Laura Castilla-Vallmanya
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DOI: dx.doi.org/10.17504/protocols.io.36wgqdb83vk5/v1
Protocol Citation: anita.adami, Laura Castilla-Vallmanya 2025. CUT and RUN - Fresh cells. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqdb83vk5/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: 117990
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 protocol describes how to perform CUT&RUN on fresh iPSC cells (optimisation might be required for other cell types).
Harvest and preparation of fresh cells
Harvest and preparation of fresh cells
5m
5m
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. Handle them carefully to avoid stress.
Pellet cell by centrifuging them at 400xg for Duration00:05:00 .

5m
Resuspend in media, count cells and harvest 500 000 cells per condition (antibody).

EXAMPLE: If you are running a CRISPRi experiment (CRISPRi gRNA hiPSCs & non-targeting control hiPSCs) and you want to perform H3K9me3 CUT&RUN on both conditions, you will harvest 500 000 CRISPRi and 500 000 control hiPSCs. Since you also need to include CUT&RUN controls (e.g. IgG control), you will need 500 000 more per condition- In total: 1 000 000 CRISPRi hiPSCs & 1 000 000control hiPSCs.

CUT&RUN
CUT&RUN
1h 53m
1h 53m
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 600xg for Duration00:03:00 .

3m
Remove supernatant, and resuspend cells in Amount800 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).

Repeat washing steps 5 & 6 2 more times.
Add Amount10 µL of pre-activated ConA-coated beads per condition (i.e. 30ul if using 3 different antibodies)

Bind cells to beads for Duration00:10:00 at RT on and end-over-end rotator, and then split bead-bound cells into three equal volumes (corresponding to IgG control, H3K4me3 and H3K9me3 treatments).

10m
Remove wash buffer and resuspend nuclei in Amount100 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 Temperature4 °C DurationOvernight 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 Temperature4 °C for Duration01:00:00 . Wash nuclei twice, resuspend in Amount100 µL digitonin buffer, and chill to Temperature0 °C -Temperature2 °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 Amount100 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 Duration00:30:00 at Temperature37 °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).