REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-H3K9me3 (dilution 1:50)	Abcam	Cat#ab8898; RRID: AB_306848
Goat anti-Rabbit IgG AF647 (dilution 1:250)	Thermo Fisher Scientific	Cat#A-21244
Chemicals, peptides, and recombinant proteins
DMSO	Sigma-Aldrich	Cat#D2650
Actinomycin D (ActD)	Sigma-Aldrich	Cat#A9415
Dulbecco’s Modified Eagle Medium (DMEM)	Thermo Fisher Scientific	Cat#41965039
Fetal bovine serum (FBS)	Thermo Fisher Scientific	Cat#10270106
Penicillin-Streptomycin (Pen Strep)	Thermo Fisher Scientific	Cat#15140122
GlutaMAX	Thermo Fisher Scientific	Cat#35050038
Dulbecco’s Phosphate Buffered Saline (DPBS)	Thermo Fisher Scientific	Cat#14190094
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	Cat#25200056
HEPES	Sigma-Aldrich	Cat#H4034
Hydrochloric acid fuming 37% (HCl)	Millipore	Cat#100317
L-Ascorbic acid	Sigma-Aldrich	Cat#A92902
Amino guanidine	Sigma-Aldrich	Cat#396494
Glucose oxidase	Sigma-Aldrich	Cat#G2133
Catalase	Sigma-Aldrich	Cat#C100
Cysteamine (MEA)	Sigma-Aldrich	Cat#30070
CuSO4	Sigma-Aldrich	Cat#C1297
Glucose	Sigma-Aldrich	Cat#G8270
5-Ethynyl-2′-deoxycytidine (EdC)	Sigma-Aldrich	Cat#T511307
Bovine serum albumin (BSA)	Sigma-Aldrich	Cat#A7906
Paraformaldehyde 16% methanol-free (PFA)	Alfa Aesar	Cat#43368
4′,6-Diamidino-2-phenylindole (DAPI)	Roche	Cat#10236276
Alexa Fluor™ 647 Azide, Triethylammonium Salt (AF647)	Thermo Fisher Scientific	Cat#A10277
Drierite	Sigma-Aldrich	Cat#238961
Deposited data
Images files (Mendeley data)	This study	DOI: https://doi.org/10.17632/pbnc4zhcwd.1
Experimental models: Cell lines
HeLa cells (human cervix epithelioid carcinoma, female)	Maria Pia Cosma Lab	N/A
Software and algorithms
ImageJ (2.0.0 and 2.1.0/1.53c)	(Rueden et al., 2017)	https://imagej.nih.gov/ij/download.html
MATLAB	MathWorks	https://www.mathworks.com/products/matlab.html
Data S1. CV analysis, related to steps 14–17, containing CV-Part1-ImageJ (steps 14–16) and CV-Part2-MATLAB (step 17)	This paper	https://github.com/CosmaLab/CV-Analysis All processing scripts can be found also in Data S1.
Data S2. Black Space analysis, related to step 18, used for the quantification of DNA-free areas	This paper	https://github.com/CosmaLab/Black-Space-Analysis All processing scripts can be found also in Data S2.
Data S3. Edge analysis, related to step 19, used for the quantification of the percentage of H3K9me3 signal at nuclear edges	This paper	https://github.com/CosmaLab/Edge-Analysis All processing scripts can be found also in Data S3.
Step-by-step simplified tutorial videos.mp4, a digest version of the protocol	This paper	STEP BY STEP simplified tutorial videos.mp4
LAS X	Leica	https://www.leica-microsystems.com/products/microscope-software/details/product/leica-las-x-ls/
NIS software STORM module	Nikon	https://www.microscope.healthcare.nikon.com/products/software/nis-elements
Graphpad Prism	GraphPad	https://www.graphpad.com/scientific-software/prism/
Insight3	(Huang et al., 2008)	N/A
Other
Borosilicate glass bottom 8-well chambers (Lab-Tek)	Thermo Fisher Scientific	Cat#155411
N-STORM 4.0 microscope equipped with highly inclined and laminated optical sheet illumination (HILO)	Nikon	N/A
LU-NV laser unit with 405 nm (LD, 20 mW output), 488 nm (DPSS, 45 mW output), 560 nm (DPSS, 45 mW output), and 647 nm (fiber, 125 mW output) lasers	Nikon	N/A
405/488/561/647nm Laser Quad Band Set	Chroma	Cat#TRF89902
CFI HP Apochromat TIRF 100XC Oil (NA 1.49) objective	Nikon	N/A
iXON Ultra DU-897U EMCCD camera	Andor Technology	N/A
Leica TCS SP8 confocal microscopy equipped with laser/filter sets to acquire DAPI signal	Leica	N/A
HC Plan-Apochromat CS2 63x/1.40 oil objective	Leica	N/A
Type F Immersion liquid (RI 1.518)	Leica	Cat#11944399
Immersion oil (Type F) (RI 1.515)	Nikon	Cat#MXA22168
Stericup Quick Release Millipore Express PLUS 0.22 μm PES, 500 mL	Millipore	Cat#11290
Telstar™ Soporte de mesa para Bio II Advance 4	Telstar	Cat#15138614
BINDER CO2 incubator CB 160, with O2 control	Binder	Cat# 9040-0092
WHEATON® Dry-Seal vacuum desiccator	Sigma-Aldrich	Cat#Z114375
Sartorius Stedim Biotech SI-215D Analytical Balance	Denver Instrument	Cat#EW-11422-04
Fisherbrand™ accumet™ AE150 Benchtop pH Meter	Thermo Fisher Scientific	Cat#13-636-AE152
Millipore Milli-Q Synthesis A10 Water Purification	Millipore	Cat#15882