TITLE: Performing CUT&RUN on adherent cells in a multi-well cell culture plate
AUTHORS: Michi Miura

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes a variant of the standard CUT&RUN procedure (Skene et al. </span><span style = "font-style:italic;">eLife</span><span> 2017; Skene et al. </span><span style = "font-style:italic;">Nature Protocols</span><span> 2018; Meers et al. </span><span style = "font-style:italic;">eLife</span><span> 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.  </span></div></div>

[STEPS]
?. [Cell seeding]
Seed adherent cells in a standard 24-well cell culture plateSeed the cells at an appropriate density so that the cells are 70 to 90% confluent when performing CUT&RUN.
?. [CUT&RUN]
Perform CUT&RUNBefore cell permeabilisation (step 2.2),Remove the cell culture mediumWash the cells with PBS (Optional) Cells can be fixed prior to CUT&RUN (step 2.1)
?. [CUT&RUN]
(Optional) Cells can be fixed by one of the followings:(1) Fix intact cellsAdd 500 μl of 1.5% formaldehyde (diluted in PBS) to a well and incubate for 10 minWash cells ×3 with PBSgo to step 2.2 (Permeabilisation)(2) Fix extracted cellsAdd 500 μl Perm buffer to a well and incubate for 5 minWash cells ×2 with PBSAdd 500 μl of 1.5% formaldehyde (diluted in PBS) to a well and incubate for 10 minWash cells ×3 with PBSgo to step 2.2 (Permeabilisation)(2) Fix extracted cellsAdd 500 μl Perm buffer to a well and incubate for 5 minWash cells ×2 with PBSAdd 500 μl of 1.5% formaldehyde (diluted in PBS) to a well and incubate for 10 minWash cells ×3 with PBSgo to step 2.2 (Permeabilisation)
?. [CUT&RUN]
PermeabilisationDispense 500 μl Perm buffer per well and incubate on the bench (i.e. at room temperature) for 15 minRemove the buffer and wash the cells once with Perm buffer
?. [CUT&RUN]
Antibody incubationPrepare antibody dilution* in Perm buffer (150 μl per well)Dispense 150 μl of the diluted antibody to a well and incubate on the bench for an hourRemove 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).
?. [CUT&RUN]
pAG-MNase incubationPrepare pAG-MNase dilution (Cell Signaling, 40366; 1:33 volume) in 150 μl Perm buffer per wellDispense 150 μl of the diluted pAG-MNase into a well and incubate on the bench for an hourRemove 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).
?. [CUT&RUN]
Chromatin digestionPrepare Perm buffer containing 5 mM CaCl2 (add 1/20 volume of 100 mM CaCl2 to Perm buffer) and chill it on iceMake sure that the cells are chilled on ice-cold waterDispense 150 μl cold Perm buffer containing 5 mM CaCl2 and incubate the plate on ice-cold water for 30 min
?. [CUT&RUN]
Fragment releaseAdd 50 μl 4× STOP solution to a well (50 μl per well)Gently rock the plate to mix the solutionPlace the cell culture plate in an incubator at 37ºC for 30 minCollect the supernatant (∼200 μl)
?. [CUT&RUN]
Fragment purificationIf 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).