Jul 14, 2020

Public workspacePerforming CUT&RUN on adherent cells in a multi-well cell culture plate

DOI
dx.doi.org/10.17504/protocols.io.bijakcie
  • Michi Miura1
  • 1Department of Microbiology, The University of Hong Kong
Michi Miura
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DOI: dx.doi.org/10.17504/protocols.io.bijakcie
External link: https://www.biorxiv.org/content/10.1101/2020.07.07.191478v1
Protocol Citation: Michi Miura 2020. Performing CUT&RUN on adherent cells in a multi-well cell culture plate. protocols.io https://dx.doi.org/10.17504/protocols.io.bijakcie
Manuscript citation:
Miura M, Chen H. CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites. Chromosome Res. 2020 Dec;28(3-4):381-393. doi: 10.1007/s10577-020-09643-0. Epub 2020 Oct 18. PMID: 33070289.
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: July 13, 2020
Last Modified: July 14, 2020
Protocol Integer ID: 39234
Keywords: CUT&RUN,
Abstract
This protocol describes a variant of the standard CUT&RUN procedure (Skene et al. eLife 2017; Skene et al. Nature Protocols 2018; Meers et al. eLife 2019). This streamlined protocol is immediately applicable to adherent cells maintained in a multi-well cell culture plate. Trypsinising cultured cells to harvest and attaching them onto Concanavalin A-beads are not required.
Guidelines
In the manuscript "CUT&RUN reveals unique positioning of pre-initiated RNA polymerase II in the steady state of transcription", this protocol was performed on the human lung carcinoma cell line A549 cultured in a 24-well polystyrene plate (https://www.biorxiv.org/content/10.1101/2020.07.07.191478v1). The protocol is expected to work for other adherent cells that are firmly attached to the plate.

Antibodies used in the above manuscript:
Millipore, 05-623 (mouse anti-RNA polymerase II, clone CTD4H8; 0.75 μg per well)
Abcam, ab5095 (rabbit anti-RNA polymerase II (phospho S2), polyclonal; 0.75 μg per well)
Abcam, ab5131 (rabbit anti-RNA polymerase II (phospho S5), polyclonal; 0.675 μg per well)

Recombinant pAG-MNase:
Purified recombinant pAG-MNase from two commercial venders were tested, of which only one from Cell Signaling (Product# 40366) worked well with this protocol.
Materials
Recombinant pAG-MNase (Cell Signaling, 40366)
Before start
Perm buffer
20 mM HEPES-KOH pH 7.5
150 mM NaCl
0.5 mM Spermidine (Sigma, S2626)
0.1% Triton X-100
Proteinase inhibitor (Roche, 04 693 132 001)

4× STOP buffer
680 mM NaCl
40 mM EDTA (Sigma, E5134)
8 mM EGTA (AG Scientific, E-2491)
100 μg/ml RNase A (Invitrogen, 12091021)
0.1% Triton X-100

Other materials
Recombinant pAG-MNase (Cell Signaling, 40366)
Spike-in DNA (Cell Signaling, 40366) can be supplemented at step 2.6 if necessary (10 pg per well).
Cell seeding
Cell seeding
Seed adherent cells in a standard 24-well cell culture plate

Seed the cells at an appropriate density so that the cells are 70 to 90% confluent when performing CUT&RUN.
CUT&RUN
CUT&RUN
Perform CUT&RUN

Before cell permeabilisation (step 2.2),
  1. Remove the cell culture medium
  2. Wash the cells with PBS

(Optional) Cells can be fixed prior to CUT&RUN (step 2.1)
(Optional) Cells can be fixed by one of the followings:

(1) Fix intact cells
  1. Add 500 μl of 1.5% formaldehyde (diluted in PBS) to a well and incubate for 10 min
  2. Wash cells ×3 with PBS
  3. go to step 2.2 (Permeabilisation)

(2) Fix extracted cells
  1. Add 500 μl Perm buffer to a well and incubate for 5 min
  2. Wash cells ×2 with PBS
  3. Add 500 μl of 1.5% formaldehyde (diluted in PBS) to a well and incubate for 10 min
  4. Wash cells ×3 with PBS
  5. go to step 2.2 (Permeabilisation)
Permeabilisation

  1. Dispense 500 μl Perm buffer per well and incubate on the bench (i.e. at room temperature) for 15 min
  2. Remove the buffer and wash the cells once with Perm buffer
Antibody incubation

  1. Prepare antibody dilution* in Perm buffer (150 μl per well)
  2. Dispense 150 μl of the diluted antibody to a well and incubate on the bench for an hour
  3. Remove the antibody solution from the well and wash the cells ×2 with Perm buffer

*1:100 dilution for antibodies of 0.5 μg/μl stock concentration and 1:200 for antibodies of 1 μg/μl (https://www.biorxiv.org/content/10.1101/2020.07.07.191478v1).
pAG-MNase incubation

  1. Prepare pAG-MNase dilution (Cell Signaling, 40366; 1:33 volume) in 150 μl Perm buffer per well
  2. Dispense 150 μl of the diluted pAG-MNase into a well and incubate on the bench for an hour
  3. Remove the pAG-MNase solution from the well and wash the cells ×2 with Perm buffer*

*At the second wash, place the cell culture plate on ice-cold water to get ready for the chromatin digestion (step 2.5).
Chromatin digestion

  1. Prepare Perm buffer containing 5 mM CaCl2 (add 1/20 volume of 100 mM CaCl2 to Perm buffer) and chill it on ice
  2. Make sure that the cells are chilled on ice-cold water
  3. Dispense 150 μl cold Perm buffer containing 5 mM CaCl2 and incubate the plate on ice-cold water for 30 min
Fragment release

  1. Add 50 μl 4× STOP solution to a well (50 μl per well)
  2. Gently rock the plate to mix the solution
  3. Place the cell culture plate in an incubator at 37ºC for 30 min
  4. Collect the supernatant (∼200 μl)
Fragment purification

If the cells were not fixed, purify the DNA fragment using DNeasy Blood & Tissue Kit (Qiagen, 69504).

If fixed (step 2.1), add 10 μl of 20% SDS to the supernatant (1% in final concentration) and incubate overnight at 65ºC. Then add Proteinase K and incubate at 56ºC for an hour. Purify the DNA with QIAquick PCR Purification Kit (Qiagen, 28104).