idr0156-study.txt

# FILL IN AS MUCH INFORMATION AS YOU CAN.  HINTS HAVE BEEN PUT IN SOME FIELDS AFTER THE HASH # SYMBOL. REPLACE THE HINT WITH TEXT WHERE APPROPRIATE.
# STUDY DESCRIPTION SECTION
# Section with generic information about the study including title, description, publication details (if applicable) and contact details

Comment[IDR Study Accession]	idr0156
Study Title	Regulation of single-cell genome organization into TADs and chromatin nanodomains
Study Type	epigenetic process
Study Type Term Source REF	NCIT
Study Type Term Accession	NCIT_C21051
Study Description	The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs). However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells,  yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation.  Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms:  cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts.  Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin,  whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale.
Study Key Words	mouse Embryonic Stem Cell (ESC)	neural progenitor cell (NPC)	chromosomes	nanoscale organization	topologically associating domain (TAD)	chromatin nanodomain (CND)	FISH	Oligopaint probes	Structured Illumination Microscopy (SIM)	single-cell analysis	CTCF	cohesin/RAD21	TSA
Study Organism	
Study Organism Term Source REF	NCBITaxon
Study Organism Term Accession	
Study Experiments Number	7
Study External URL	
Study BioImage Archive Accession
Study Public Release Date	2025-05-08

# Study Publication
Study PubMed ID	33077913
Study Publication Title	Regulation of single-cell genome organization into TADs and chromatin nanodomains
Study Author List	Szabo Q, Donjon A, Jerkovic I, Papadopoulos GL, Cheutin T, Bonev B, Nora EP, Bruneau BG, Bantignies F, Cavalli G
Study PMC ID	PMC7610512
Study DOI	https://doi.org/10.1038/s41588-020-00716-8

# Study Contacts
Study Person Last Name	Szabo	Bantignies	Cavalli	Mateos-Langerak
Study Person First Name	Quentin	Frédéric	Giacomo	Julio
Study Person Email	quentin.szabo@uzh.ch	frederic.bantignies@igh.cnrs.fr	giacomo.cavalli@igh.cnrs.fr	julio.mateos-langerak@igh.cnrs.fr
Study Person Address	University of Zurich, Department of Molecular Life Sciences
Zürich, Switzerland	Institute of Human Genetics,  Centre National de la Recherche Scientifique, University of Montpellier, Montpellier, France.	Institute of Human Genetics,  Centre National de la Recherche Scientifique, University of Montpellier, Montpellier, France.	IGH, University of Montpellier, CNRS, 141 rue de la Cardonille, 34396 Montpellier, France.  Montpellier Ressources Imagerie, BioCampus, University of Montpellier, CNRS, INSERM, 141 rue de la Cardonille, 34094 Montpellier, France.
Study Person ORCID	0000-0002-3539-7875	0000-0003-4063-7324	0000-0003-3709-3469	0000-0003-1579-0773
Study Person Roles	First author	Corresponding author	Corresponding author	IDR deposition

# Study License and Data DOI
Study License	CC BY 4.0
Study License URL	https://creativecommons.org/licenses/by/4.0/
Study Copyright	Szabo et al
Study Data Publisher	University of Dundee
Study Data DOI	https://doi.org/10.17867/10000206	

Term Source Name	NCBITaxon	EFO	CMPO	FBbi
Term Source URI	http://purl.obolibrary.org/obo/	http://www.ebi.ac.uk/efo/	http://www.ebi.ac.uk/cmpo/,http://purl.obolibrary.org/obo/

# EXPERIMENT SECTION
# Experiment Section containing all information relative to each experiment in the study including materials used, protocols names and description, phenotype names and description. For multiple experiments this section should be repeated.  Copy and paste the whole section below and fill out for the next experiment

Experiment Number	1
Comment[IDR Experiment Name]	idr0156-szabo-tadcnd/experimentA
Experiment Data DOI	https://doi.org/10.17867/10000206a
Experiment Sample Type	cell
Experiment Description	Mouse embryonic stem cells (ESCs) were cultured in the conditions described below (see Protocol Description) and they were fixed on coverslips prior to the Fluorescent in situ Hybridization (DNA FISH) procedure. A large set of Oligopaint probes covering Topologically Associating Domains (TADs) of various size and various epigenetic signatures were used for the DNA FISH. Coverslips with hybridized cells were then observed with Structured Illumination Microscopy (3D-SIM). This approach allows for the description of TAD organization in mouse ESCs (Overlap Fractions (OFs) and 3D distances with consecutive Oligopaint probes within TADs or between TADs, see the article) and the identification of TAD sub-structures, dubbed Chromatin NanoDomains (CNDs), by applying watershed-segmentation on the Oligopaint probe fluorescent signal. Assays TSA and TSA-CTL (control) were performed in parallel with and without trichostatin-A (TSA), respectively. TSA is a potent inhibitor of histone deacetylases that induces histone hyperacetylation.
Experiment Size 5D Images: 	Average Image Dimension (XYZCT): 55x55x56xCx1	Total Tb: 0.002
Experiment Example Images	20180814_RI510_ESC_51b_51a_SIR_2C_ALN_THR_20.ome.tiff
Experiment Imaging Method	structured illumination microscopy (SIM)
Experiment Imaging Method Term Source REF	Fbbi
Experiment Imaging Method Term Accession	FBbi_00000332
Experiment Organism	Mus musculus
Experiment Organism Term Source REF	NCBITaxon
Experiment Organism Term Accession	10090
Experiment Comments	3D-SIM.  A total of 26 Oligopaint probes (11, 12, 21, 22, 32, 33, 51, 51a, 51b, 52, 61a, 61b, 62, 81, 82a, 82b, 101, 102a, 102b, 103, 111, 112, 121a, 121b, 141a, 141b) were monitored in the ESC assay. Most of the probes were monitored by pair to measure their intermingling (consecutive probes within a given TAD) or to measure their partitioning along the genome (consecutive probes of two adjacent TADs, separated by a TAD border). Refer to Extented Data Fig.1 of the article to visualize the probes on specific Hi-C maps. For example, in ESC, probes 11 and 12 or 51a and 51b are two consecutive probes belonging to the same TAD, probes 32 and 33 or 51 and 52 are two consecutive probes belonging to two consecutive TADs, therefore separated by a TAD border. ESC_TSA and ESC_TSA-CTL assays were performed using 11 Oligopaint probes (32, 62, 81, 101, 102a, 102b, 103, 111, 112, 121a, 121b).

# assay files
Experiment Assay File	idr0156-experimentA-annotation.csv
Experiment Assay File Format	tab-delimited text
Assay Experimental Conditions	Mouse standard E14 cell line (E14Tg2a.4) were used as the wild-type ESC control.
Assay Experimental Conditions Term Source REF
Assay Experimental Conditions Term Accession
Quality Control Description	Mouse standard E14 cell line (E14Tg2a.4) were purchased directly from MMRRC, UC Davis, and were not re-authenticated besides the presence of the Oct4 ESC marker (monitored by RTqPCR and immuno-labeling using an anti-Oct4 antibody (catalog no. 5677S; Cell signaling)). The successful hybridization of oligopaints recognizing mouse-specific sequences to the genome is another quality control for the nature of the cells.

# Protocols
Protocol Name	Mouse ESCs standard culture	No treatment or TSA treatment	Oligopaint FISH	Otsu's method and watershed segmentation
Protocol Type	Cell culture	No treatment or TSA treatment	DNA FISH	Image segmentation
Protocol Type Term Source REF	EFO	EFO	EFO
Protocol Type Term Accession	EFO_0003789	EFO_0003969	EFO_0011016
Protocol Description	Mouse ESCs (E14Tg2a.4 cell line, MMRRC, UC Davis) were cultured on 0.1% gelatin-coated (catalog no.G1890-100G; Sigma-Aldrich) dishes in Glasgow's MEM (catalog no. 21710025; Gibco), supplemented with 15% FCS qualified serum (catalog no. 26140079, US origin; Thermo Fisher Scientific), 1x GlutaMAX (catalog no. 35050038; Thermo Fisher Scientific), 1x MEM non-essential amino acids solution (catalog no.11140035; Thermo Fisher Scientific), 50 U penicillin-streptomycin (catalog no.15140122; Gibco), 0.1 mM sodium pyruvate (catalog no. 11360070; Gibco), 0.1 mM 2-mercaptoethanol (catalog no. 31350010; Gibco) and 1,000 U ml-1 recombinant mouse LIF protein (catalog no. ESG1107; Sigma-Aldrich). The medium was changed every day and cells were passaged every 2 days using the TrypLE express enzyme (catalog no. 12604013; Gibco). Two days before the FISH, approximately 150.000 cells were plated in a 6-well plate containing an autoclaved glass coverslip (170 ± 5 μm (ZEISS)) coated with 0.1% gelatin, with fresh medium replacement after one day. Mouse ESCs were grown on the coverslip for a total of 2 days and washed once with PBS before fixation prior to the FISH procedure. For TSA treatment, the day of the collection, medium was replaced with fresh medium containing TSA (final concentration at 100 ng/ml) (catalog no. T-8552; Sigma-Aldrich) for 6h before collecting cells for the FISH procedure.	Standard growth conditions. If no specific treatment, use as the control condition. TSA treatment for 6h (see details of the protocol on the left column).	Cells were hybridized with the Oligopaint fluorescent probes using a DNA FISH protocol, which is briefly described in the article and a detailed protocol can be found in Szabo et al. (PMID: 32820407; DOI: 10.1007/978-1-0716-0664-3_13). Here, pairs of Oligopaint probes labeled with Alexa Fluor 488 and ATTO 565 fluorophores were used for OFs and 3D distance measurements, only Oligopaint probes labeled with ATTO 565 were used to count the number of  CNDs inside TADs. The number of oligos per Oligopaint probe depends on its genomic size and the density of oligos found in that region. The coordinates, the size and the number of oligos for each Oligopaint probe can be found in the Extended Data Fig.9 of the article. Each oligo carryies 3 fluorophores allowing a robust fluorescent signal of each Oligopaint probe. Nuclei were counterstained with DAPI. Coverslips containing cells were mounted on slides with VECTASHIELD and sealed with nail polish. 3D-SIM imaging was performed with a DeltaVision OMX V4 microscope equipped with a x100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (Evolve 512B; Photometrics) camera for a pixel size of 80 nm. Diode lasers at 405, 488, 561 and 647 nm were used with the standard corresponding emission filters. Z-stacks were acquired by scanning the sample in the axial direction (z-step of 125 nm) using 5 phases and 3 angles per image plane. Raw images were reconstructed using SoftWorx v.6.5 (GE Healthcare Systems) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). The quality of reconstructed images was assessed using the SIMcheck plugin (doi: 10.1038/srep15915) of ImageJ v.1.52i.	Multiple channel images were aligned using Chromagnon (doi: 10.1038/s41598-018-25922-7). Images were smoothed using 3D Gaussian filters (σ = 0.5), and FISH probes were segmented in 3D using Otsu’s method. Segmented objects smaller than 0.04 μm3 or in contact to the image border were discarded. Images with more than one segmented object per channel were discarded. For CND quantification, the watershed function was applied to the complement of the scaled intensity values (0 to 1, without Gaussian filter) within ROIs defined by the full probe segmentations described above. Watershed segmented objects smaller than 0.0072 μm3 were discarded.

# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession

# Feature Level Data Files (give individual file details unless there is one file per well)
Feature Level Data File Name
Feature Level Data File Format
Feature Level Data File Description
Feature Level Data Column Name
Feature Level Data Column Description

#  Processed Data Files
Processed Data File Name	idr0156-experimentA-processed.csv
Processed Data File Format	tab-delimited text
Processed Data File Description	Key measurements on the segmented domains, subdomains and overlapping regions.
Processed Data Column Name	Image Name	Channel ID	roi_type	label	area	major_axis_length	centroid-0	centroid-1	centroid-2	weighted_centroid-0	weighted_centroid-1	weighted_centroid-2	equivalent_diameter	max_intensity	mean_intensity	min_intensity	sphericity	solidity	volume	volume_units	overlap_fraction	distance_x	distance_y	distance_z	distance3d	distance_units	Roi Name
Processed Data Column Type	linking column	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data
Processed Data Column Annotation Level	Image	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi
Processed Data Column Description	Name of the image file	Id of the channel on which the measurements were taken. Empty where referring to overlapping regions	The type of segmented region: domain, subdomain or overlap	The label of the roi in the corresponding rois files	The volume of the segmented region in voxels	The length of the major axis of the region	Z-position of the centroid of the region	Y-position of the centroid of the region	X-position of the centroid of the region	Z-position of the center of mass of the region	Y-position of the center of mass of the region	X-position of the center of mass of the region	The diameter of a circle with the same area as the region	Max intensity in the region	Mean intensity in the region	Min intensity in the region	The ratio of the area of a sphere with the same volume as the region to the actual surface area of the region	Ratio of pixels in the region to pixels of the convex hull image	Volume of the region in physical units	Units of the volume	Fraction of the two domain that is overlapping	Distance between the centers of mass of the two domains in the x dimension	Distance between the centers of mass of the two domains in the y dimension	Distance between the centers of mass of the two domains in the z dimension	Distance between the centers of mass of the two domains in 3D	Units of the distances	name of the ROI
Processed Data Column Link To Assay File	Image Name

Experiment Number	2
Comment[IDR Experiment Name]	idr0156-szabo-tadcnd/experimentB
Experiment Data DOI	https://doi.org/10.17867/10000206b
Experiment Sample Type	cell
Experiment Description	Mouse neural progenitor cells (NPCs) were obtained in the conditions described below (see Protocol Description) and they were fixed on coverslips prior to the Fluorescent in situ Hybridization (DNA FISH) procedure. A large set of Oligopaint probes covering Topologically Associating Domains (TADs) of various size and various epigenetic signatures were used for the DNA FISH. Coverslips with hybridized cells were then observed with Structured Illumination Microscopy (3D-SIM). This approach allows for the description of TAD organization in mouse NPCs (Overlap Fractions (OFs) and 3D distances with consecutive Oligopaint probes within TADs or between TADs, see the article) and the identification of TAD sub-structures, dubbed Chromatin NanoDomains (CNDs), by applying watershed-segmentation on the Oligopaint probe fluorescent signal. These data were compared to the ESC conditions from Experiment Mouse_ESC_TADs. NPC from cryo-section of mouse brain neocortex of  E14.5 C57BL/6J embryos (ncxNPC) were subjected to the DNA FISH procedure as in Experiment Mouse_ESC_TADs. Two pairs of TAD probes were observed and were monitored by pair to compare their degree of intermingling (Overlap Fractions (OFs) and 3D distances with consecutive Oligopaint probes between adjacent TADs, see the article) with ESCs from Experiment Mouse_ESC_TADs and this experiment. Slides with hybridized cells were observed with 3D-SIM.
Experiment Size 5D Images: 	Average Image Dimension (XYZCT): 55x55x56xCx1	Total Tb: 0.001
Experiment Example Images
Experiment Imaging Method	structured illumination microscopy (SIM)
Experiment Imaging Method Term Source REF	Fbbi
Experiment Imaging Method Term Accession	FBbi_00000332
Experiment Organism	Mus musculus
Experiment Organism Term Source REF	NCBITaxon
Experiment Organism Term Accession	10090
Experiment Comments	3D-SIM.  In the NPC dataset, a total of 26 Oligopaint probes (11, 12, 21, 22, 32, 33, 51, 51a, 51b, 52, 61a, 61b, 62, 81, 82a, 82b, 101, 102a, 102b, 103, 111, 112, 121a, 121b, 141a, 141b) were monitored in this experiment. Most of the probes were monitored by pair to measure their intermingling (consecutive probes within a given TAD) or to measure their partitioning along the genome (consecutive probes of two adjacent TADs, separated by a TAD border). Refer to Extented Data Fig.1 of the article to visualize the probes on specific Hi-C maps. For example, in NPC, probes 21 and 22 or 51a and 52b are two consecutive probes belonging to the same TAD, probes 51 and 52 are two consecutive probes belonging to two consecutive TADs, separated by a TAD border. In the ncxNPC dataset, a total of 4 Oligopaint probes (11, 12, 21, 22) were monitored.

# assay files
Experiment Assay File	idr0156-experimentB-annotation.csv
Experiment Assay File Format	tab-delimited text
Assay Experimental Conditions	Mouse NPCs differentiated from standard E14 cell line (E14Tg2a.4) detailed in Experiment Mouse_ESC_TADs.
Assay Experimental Conditions Term Source REF
Assay Experimental Conditions Term Accession
Quality Control Description	Mouse NPCs were obtained from differentiation of ESCs described in Experiment Mouse_ESC_TADs. The presence of the Pax6 NPC marker was verified by RTqPCR and immuno-labeling using an anti-Pax6 (catalog no. PRB-278P-0100; Covance). In the tissue sections (ncxNPC), NPC were localized according to their position in the brain neocortex, i.e. in the ventricular zone containing apical progenitors (Pax6 positive NPC) (Bonev et al., doi: 10.1016/j.cell.2017.09.043). All mice were housed and maintained according to the guidelines of the Animal Care and Use Committee of the Institut National de la Santé et de la Recherche Médicale (INSERM) in accordance with the European Council directive 2010/63/UE for the protection and use of vertebrate animals.

# Protocols
Protocol Name	Mouse ESCs standard culture and differentiation into NPCs	NPC differentiation	Oligopaint FISH	Otsu's method and watershed segmentation
Protocol Type	Cell culture	Treatment for NPC differentiation	DNA FISH	Image segmentation
Protocol Type Term Source REF	EFO	EFO	EFO
Protocol Type Term Accession	EFO_0003789	EFO_0003969	EFO_0011016
Protocol Description	In the NPC dataset, mouse ESCs described in Experiment Mouse_ESC_TADs were differentiated into NPCs as described by Gaspard et al. (doi: 10.1038/nprot.2009.157) with the following changes. Cells were plated (2.5 x 10e5 cells per 10-cm dish) on 0.1% gelatin-coated dishes in the ESC medium described in Experiment Mouse_ESC_TADs. After 1 day, cells were cultured in DDM medium (DMEM/F-12, GlutaMAX supplement; catalog no. 31331028; Gibco), supplemented with 0.05% bovine albumin fraction (catalog no. 15260037; Gibco), 1x MEM non-essential amino acids, 50 U penicillin-streptomycin, 1x N-2 supplement (catalog no. 17502048; Gibco), 1 mM sodium pyruvate and 0.1 mM 2-mercaptoethanol. From day 2 to day 10, medium was changed every 2 days and cyclopamine V. californicum (catalog no. 239803; Merck Millipore) was added to the medium (final concentration of 0.4ug/ml). After 12 days of differentiation, cells were dissociated with StemPro Accutase (catalog no. A1110501; Gibco), plated on 6-well plates (containing an autoclaved glass coverslip (170 ± 5 μm (ZEISS)) coated with poly-L-lysine (catalog no. P2636; Sigma-Aldrich)/laminin (catalog no. 11243217001; Sigma-Aldrich) and cultured in a 1:1 mixture of DDM and neurobasal medium (catalog no. 21103049; Gibco) supplemented with 1X B-27 supplement minus vitamin A (catalog no. 12587010; Gibco) and 1x GlutaMAX supplement and 50 U penicillin-streptomycin for an additional 2 days (day 12 + 2) to obtain NPCs. Coverslips containing NPCs were then washed once with PBS before fixation prior to the FISH procedure. For the ncxNPC dataset, Mouse brains from E14.5 C57BL/6J embryos were dissected and fixed in 4% paraformaldehyde (PFA) overnight at 4°C. They were then cryopreserved for 24 h at 4°C in a PBS-20% sucrose solution. After embedding in optimal cutting temperature compound and rapid freezing in isopentane, brains were sectioned at 10-um width on glass slides with a cryostat (Leica Biosystems). Slides containing fixed mouse brain neocortex were then submitted to the FISH procedure.	For NPC differentiation conditions and ncxNPC preparation (see details of the protocol on the left column).	Cells were hybridized with the Oligopaint fluorescent probes using a DNA FISH protocol, which is briefly described in the article and a detailed protocol can be found in Szabo et al. (PMID: 32820407; DOI: 10.1007/978-1-0716-0664-3_13). Here, pairs of Oligopaint probes labeled with Alexa Fluor 488 and ATTO 565 fluorophores were used for OFs  and 3D distance measurements, only Oligopaint probes labeled with ATTO 565 were used to count the number of  CNDs inside TADs. The number of oligos per Oligopaint probe depends on its genomic size and the density of oligos found in that region. The coordinates, the size and the number of oligos for each Oligopaint probe can be found in the Extended Data Fig.9 of the article. Each oligo carryies 3 fluorophores allowing a robust fluorescent signal of each Oligopaint probe. Nuclei were counterstained with DAPI. Coverslips containing cells were mounted on slides with VECTASHIELD and sealed with nail polish. 3D-SIM imaging was performed with a DeltaVision OMX V4 microscope equipped with a x100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (Evolve 512B; Photometrics) camera for a pixel size of 80 nm. Diode lasers at 405, 488, 561 and 647 nm were used with the standard corresponding emission filters. Z-stacks were acquired by scanning the sample in the axial direction (z-step of 125 nm) using 5 phases and 3 angles per image plane. Raw images were reconstructed using SoftWorx v.6.5 (GE Healthcare Systems) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). The quality of reconstructed images was assessed using the SIMcheck plugin (doi: 10.1038/srep15915) of ImageJ v.1.52i.	Multiple channel images were aligned using Chromagnon (doi: 10.1038/s41598-018-25922-7). Images were smoothed using 3D Gaussian filters (σ = 0.5), and FISH probes were segmented in 3D using Otsu’s method. Segmented objects smaller than 0.04 μm3 or in contact to the image border were discarded. Images with more than one segmented object per channel were discarded. For CND quantification, the watershed function was applied to the complement of the scaled intensity values (0 to 1, without Gaussian filter) within ROIs defined by the full probe segmentations described above. Watershed segmented objects smaller than 0.0072 μm3 were discarded.

# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession

# Feature Level Data Files (give individual file details unless there is one file per well)
Feature Level Data File Name
Feature Level Data File Format
Feature Level Data File Description
Feature Level Data Column Name
Feature Level Data Column Description

#  Processed Data Files
Processed Data File Name	idr0156-experimentB-processed.csv
Processed Data File Format	tab-delimited text
Processed Data File Description	Key measurements on the segmented domains, subdomains and overlapping regions.  
Processed Data Column Name	Image Name	Channel ID	roi_type	label	area	major_axis_length	centroid-0	centroid-1	centroid-2	weighted_centroid-0	weighted_centroid-1	weighted_centroid-2	equivalent_diameter	max_intensity	mean_intensity	min_intensity	sphericity	solidity	volume	volume_units	overlap_fraction	distance_x	distance_y	distance_z	distance3d	distance_units	Roi Name
Processed Data Column Type	linking column	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data
Processed Data Column Annotation Level	Image	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi
Processed Data Column Description	Name of the image file	Id of the channel on which the measurements were taken. Empty where referring to overlapping regions	The type of segmented region: domain, subdomain or overlap	The label of the roi in the corresponding rois files	The volume of the segmented region in voxels	The length of the major axis of the region	Z-position of the centroid of the region	Y-position of the centroid of the region	X-position of the centroid of the region,Z-position of the center of mass of the region	Y-position of the center of mass of the region,X-position of the center of mass of the region	The diameter of a circle with the same area as the region	Max intensity in the region	Mean intensity in the region	Min intensity in the region	The ratio of the area of a sphere with the same volume as the region to the actual surface area of the region	Ratio of pixels in the region to pixels of the convex hull image	Volume of the region in physical units	Units of the volume	Fraction of the two domain that is overlapping	Distance between the centers of mass of the two domains in the x dimension	Distance between the centers of mass of the two domains in the y dimension	Distance between the centers of mass of the two domains in the z dimension	Distance between the centers of mass of the two domains in 3D	Units of the distances	Name of the ROI
Processed Data Column Link To Assay File	Image Name

Experiment Number	3
Comment[IDR Experiment Name]	idr0156-szabo-tadcnd/experimentC
Experiment Data DOI	https://doi.org/10.17867/10000206c
Experiment Sample Type	cell
Experiment Description	Same experience as in Mouse_ESC_TADs, but with mouse CTCF-AID ES cells allowing for the rapid degradation of the CTCF factor. Assays CTCF-AID_AUX and CTCF-AID_AUX-CTL (control) were performed in parallel with and without Auxin, respectively. Auxin treatment induces the rapid degradation of CTCF.
Experiment Size 5D Images: 	Average Image Dimension (XYZCT): 55x55x56xCx1	Total Tb: 0.002
Experiment Example Images
Experiment Imaging Method	structured illumination microscopy (SIM)
Experiment Imaging Method Term Source REF	Fbbi
Experiment Imaging Method Term Accession	FBbi_00000332
Experiment Organism	Mus musculus
Experiment Organism Term Source REF	NCBITaxon
Experiment Organism Term Accession	10090
Experiment Comments	3D-SIM.  A total of 25 Oligopaint probes (11, 12, 21, 22, 32, 33, 51a, 51b, 52, 61a, 61b, 62, 81, 82a, 82b, 101, 102a, 102b, 103, 111, 112, 121a, 121b, 141a, 141b) were monitored in this experiment.

# assay files
Experiment Assay File	idr0156-experimentC-annotation.csv
Experiment Assay File Format	tab-delimited text
Assay Experimental Conditions	Mouse CTCF-AID ES cells (ID no. EN204.3; doi: 10.1016/j.molcel.2019.08.015) were cultured in the same condition as in Experiment Mouse_ESC_TADs. Auxin was applied in dataset CTCF-AID_AUX.
Assay Experimental Conditions Term Source REF
Assay Experimental Conditions Term Accession
Quality Control Description	mESC E14 CTCF-GFP-Tir1-TIGRE (referred to as CTCF-AID) was originally described in Saldana-Meyer et al. (doi: 10.1016/j.molcel.2019.08.015). The parental mESC E14 used to generate mESC E14 CTCF-GFP-Tir1-TIGRE line was initially authenticated in the laboratory of Benoit Bruneau by karyotyping and genotyping with primers that detected homozygous SNPs of 129 strains, but was not re-authenticated for this study. The CTCF degradation (after AUX treatment) was verified by Western-Blot using an anti-CTCF antibody (catalog no. 61311; Active motif) and an anti-Vinculin (catalog no. sc-73614; SantaCruz) as loading control.

# Protocols
Protocol Name	Mouse ESCs standard culture	No treatment or Auxin treatment	Oligopaint FISH	Otsu's method and watershed segmentation
Protocol Type	Cell culture	No treatment or Auxin treatment for CTCF degradation	DNA FISH	Image segmentation
Protocol Type Term Source REF	EFO	EFO	EFO
Protocol Type Term Accession	EFO_0003789	EFO_0003969	EFO_0011016
Protocol Description	Mouse CTCF-AID ES cells (ID no. EN204.3; doi: 10.1016/j.molcel.2019.08.015) were cultured in the same condition as in Experiment Mouse_ESC_TADs. The induction of CTCF degradation by the Auxin-inducible degron (AID) system (referred to as AUX) was performed as described in Nora et al. (doi: 10.1016/j.cell.2017.05.004). Two days before the FISH, approximately 150.000 cells were plated in a 6-well plate containing an autoclaved glass coverslip (170 ± 5 μm (ZEISS)) coated with 0.1% gelatin, with medium containing indole-3-acetic acid sodium salt (final concentration at 500 µM) (auxin analog; catalog no. 16954; Cayman Chemical), and fresh medium-containing Auxin replacement after one day. Mouse ESCs were grown on the coverslip for a total of 2 days in the presence of Auxin and washed once with PBS before fixation prior to the FISH procedure.	No specific treatment or Auxin treatment for 2 days for CTCF degradation (see details on the left column).	Cells were hybridized with the Oligopaint fluorescent probes using a DNA FISH protocol, which is briefly described in the article and a detailed protocol can be found in Szabo et al. (PMID: 32820407; DOI: 10.1007/978-1-0716-0664-3_13). Here, pairs of Oligopaint probes labeled with ATTO 565 and ATTO 647 fluorophores were used for OFs  and 3D distance measurements, only Oligopaint probes labeled with ATTO 565 were used to count the number of  CNDs inside TADs. The number of oligos per Oligopaint probe depends on its genomic size and the density of oligos found in that region. The coordinates, the size and the number of oligos for each Oligopaint probe can be found in the Extended Data Fig.9 of the article. Each oligo carryies 3 fluorophores allowing a robust fluorescent signal of each Oligopaint probe. Nuclei were counterstained with DAPI. Coverslips containing cells were mounted on slides with VECTASHIELD and sealed with nail polish. 3D-SIM imaging was performed with a DeltaVision OMX V4 microscope equipped with a x100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (Evolve 512B; Photometrics) camera for a pixel size of 80 nm. Diode lasers at 405, 488, 561 and 647 nm were used with the standard corresponding emission filters. Z-stacks were acquired by scanning the sample in the axial direction (z-step of 125 nm) using 5 phases and 3 angles per image plane. Raw images were reconstructed using SoftWorx v.6.5 (GE Healthcare Systems) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). The quality of reconstructed images was assessed using the SIMcheck plugin (doi: 10.1038/srep15915) of ImageJ v.1.52i.	Multiple channel images were aligned using Chromagnon (doi: 10.1038/s41598-018-25922-7). Images were smoothed using 3D Gaussian filters (σ = 0.5), and FISH probes were segmented in 3D using Otsu’s method. Segmented objects smaller than 0.04 μm3 or in contact to the image border were discarded. Images with more than one segmented object per channel were discarded. For CND quantification, the watershed function was applied to the complement of the scaled intensity values (0 to 1, without Gaussian filter) within ROIs defined by the full probe segmentations described above. Watershed segmented objects smaller than 0.0072 μm3 were discarded.

# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession

# Feature Level Data Files (give individual file details unless there is one file per well)
Feature Level Data File Name
Feature Level Data File Format
Feature Level Data File Description
Feature Level Data Column Name
Feature Level Data Column Description

#  Processed Data Files
Processed Data File Name	idr0156-experimentC-processed.csv
Processed Data File Format	tab-delimited text
Processed Data File Description	Key measurements on the segmented domains, subdomains and overlapping regions.  
Processed Data Column Name	Image Name	Channel ID	roi_type	label	area	major_axis_length	centroid-0	centroid-1	centroid-2	weighted_centroid-0	weighted_centroid-1	weighted_centroid-2	equivalent_diameter	max_intensity	mean_intensity	min_intensity	sphericity	solidity	volume	volume_units	overlap_fraction	distance_x	distance_y	distance_z	distance3d	distance_units	Roi Name
Processed Data Column Type	linking column	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data
Processed Data Column Annotation Level	Image	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi
Processed Data Column Description	Name of the image file	Id of the channel on which the measurements were taken. Empty where referring to overlapping regions	The type of segmented region: domain, subdomain or overlap	The label of the roi in the corresponding rois files	The volume of the segmented region in voxels	The length of the major axis of the region	Z-position of the centroid of the region	Y-position of the centroid of the region	X-position of the centroid of the region	Z-position of the center of mass of the region	Y-position of the center of mass of the region	X-position of the center of mass of the region	The diameter of a circle with the same area as the region	Max intensity in the region	Mean intensity in the region	Min intensity in the region	The ratio of the area of a sphere with the same volume as the region to the actual surface area of the region	Ratio of pixels in the region to pixels of the convex hull image	Volume of the region in physical units	Units of the volume	Fraction of the two domain that is overlapping	Distance between the centers of mass of the two domains in the x dimension	Distance between the centers of mass of the two domains in the y dimension	Distance between the centers of mass of the two domains in the z dimension	Distance between the centers of mass of the two domains in 3D	Units of the distances	Name of the ROI
Processed Data Column Link To Assay File	Image Name

Experiment Number	4
Comment[IDR Experiment Name]	idr0156-szabo-tadcnd/experimentD
Experiment Data DOI	https://doi.org/10.17867/10000206d
Experiment Sample Type	cell
Experiment Description	Same experience as in  Mouse_ESC_TADs, but with mouse RAD21-AID ES cells allowing for the rapid degradation of RAD21, a cohesin complex subunit. Assays RAD21-AID_AUX and RAD21-AID_AUX-CTL (control) were performed in parallel with and without Auxin, respectively. Auxin treatment induces the rapid degradation of RAD21.
Experiment Size 5D Images: 	Average Image Dimension (XYZCT): 55x55x56xCx1	Total Tb: 0.003
Experiment Example Images
Experiment Imaging Method	structured illumination microscopy (SIM)
Experiment Imaging Method Term Source REF	Fbbi
Experiment Imaging Method Term Accession	FBbi_00000332
Experiment Organism	Mus musculus
Experiment Organism Term Source REF	NCBITaxon
Experiment Organism Term Accession	10090
Experiment Comments	3D-SIM.  A total of 25 Oligopaint probes (11, 12, 21, 22, 32, 33, 51a, 51b, 52, 61a, 61b, 62, 81, 82a, 82b, 101, 102a, 102b, 103, 111, 112, 121a, 121b, 141a, 141b) were monitored in this experiment.

# assay files
Experiment Assay File	idr0156-experimentD-annotation.csv
Experiment Assay File Format	tab-delimited text
Assay Experimental Conditions	Mouse RAD21-AID cells (EN272.2 cell line, this study) were cultured in the same condition as in Experiment Mouse_ESC_TADs. Auxin was applied in dataset RAD21-AID_AUX.
Assay Experimental Conditions Term Source REF
Assay Experimental Conditions Term Accession
Quality Control Description	mESC E14 RAD21-eGFP-Tir1-TIGRE (refered to as RAD21-AID) are described in this study. They were generated in the laboratory of Benoit Bruneau and are described in the methods section of the paper. The parental mESC E14 used to generate mESC E14 RAD21-eGFP-Tir1-TIGRE line was initially authenticated in the laboratory of Benoit Bruneau by karyotyping and genotyping with primers that detected homozygous SNPs of 129 strains, but were not re-authenticated for this study. The RAD21 degradation (after AUX treatment) was verified by Western-Blot using an anti-RAD21 antibody (catalog no. ab154769; Abcam) and an anti-Vinculin (catalog no. sc-73614; SantaCruz) as loading control.

# Protocols
Protocol Name	Mouse ESCs standard culture	No treatment or Auxin treatment	Oligopaint FISH	Otsu's method and watershed segmentation
Protocol Type	Cell culture	No treatment or Auxin treatment for RAD21 degradation	DNA FISH	Image segmentation
Protocol Type Term Source REF	EFO	EFO	EFO
Protocol Type Term Accession	EFO_0003789	EFO_0003969	EFO_0011016
Protocol Description	Mouse RAD21-AID cells (EN272.2 cell line, this study) were cultured in the same condition as in Experiment Mouse_ESC_TADs. The induction of RAD21 degradation by the Auxin-inducible degron (AID) system (referred to as AUX) was performed as described in this study. Two days before the FISH, approximately 150.000 cells were plated in a 6-well plate containing an autoclaved glass coverslip (170 ± 5 μm (ZEISS)) coated with 0.1% gelatin, with fresh medium replacement after one day. The day of the collection, medium was replaced with fresh medium containing indole-3-acetic acid sodium salt (final concentration at 500 µM) (auxin analog; catalog no. 16954; Cayman Chemical) for 6h. Mouse ESCs were grown on the coverslip for a total of 2 days, induced the last 6 hours with Auxin and washed once with PBS before fixation prior to the FISH procedure.	No specific treatment or Auxin treatment for 6 hours for RAD21 degradation (see details on the left column).	Cells were hybridized with the Oligopaint fluorescent probes using a DNA FISH protocol, which is briefly described in the article and a detailed protocol can be found in Szabo et al. (PMID: 32820407; DOI: 10.1007/978-1-0716-0664-3_13). Here, pairs of Oligopaint probes labeled with ATTO 565 and ATTO 647 fluorophores were used for OFs  and 3D distance measurements, only Oligopaint probes labeled with ATTO 565 were used to count the number of  CNDs inside TADs. The number of oligos per Oligopaint probe depends on its genomic size and the density of oligos found in that region. The coordinates, the size and the number of oligos for each Oligopaint probe can be found in the Extended Data Fig.9 of the article. Each oligo carryies 3 fluorophores allowing a robust fluorescent signal of each Oligopaint probe. Nuclei were counterstained with DAPI. Coverslips containing cells were mounted on slides with VECTASHIELD and sealed with nail polish. 3D-SIM imaging was performed with a DeltaVision OMX V4 microscope equipped with a x100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (Evolve 512B; Photometrics) camera for a pixel size of 80 nm. Diode lasers at 405, 488, 561 and 647 nm were used with the standard corresponding emission filters. Z-stacks were acquired by scanning the sample in the axial direction (z-step of 125 nm) using 5 phases and 3 angles per image plane. Raw images were reconstructed using SoftWorx v.6.5 (GE Healthcare Systems) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). The quality of reconstructed images was assessed using the SIMcheck plugin (doi: 10.1038/srep15915) of ImageJ v.1.52i.	Multiple channel images were aligned using Chromagnon (doi: 10.1038/s41598-018-25922-7). Images were smoothed using 3D Gaussian filters (σ = 0.5), and FISH probes were segmented in 3D using Otsu’s method. Segmented objects smaller than 0.04 μm3 or in contact to the image border were discarded. Images with more than one segmented object per channel were discarded. For CND quantification, the watershed function was applied to the complement of the scaled intensity values (0 to 1, without Gaussian filter) within ROIs defined by the full probe segmentations described above. Watershed segmented objects smaller than 0.0072 μm3 were discarded.

# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession

# Feature Level Data Files (give individual file details unless there is one file per well)
Feature Level Data File Name
Feature Level Data File Format
Feature Level Data File Description
Feature Level Data Column Name
Feature Level Data Column Description

#  Processed Data Files
Processed Data File Name	idr0156-experimentD-processed.csv
Processed Data File Format	tab-delimited text
Processed Data File Description	Key measurements on the segmented domains, subdomains and overlapping regions.  
Processed Data Column Name	Image Name	Channel ID	roi_type	label	area	major_axis_length	centroid-0	centroid-1	centroid-2	weighted_centroid-0	weighted_centroid-1	weighted_centroid-2	equivalent_diameter	max_intensity	mean_intensity	min_intensity	sphericity	solidity	volume	volume_units	overlap_fraction	distance_x	distance_y	distance_z	distance3d	distance_units	Roi Name
Processed Data Column Type	linking column	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data	data
Processed Data Column Annotation Level	Image	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi	roi
Processed Data Column Description	Name of the image file,Id of the channel on which the measurements were taken. Empty where referring to overlapping regions	The type of segmented region: domain, subdomain or overlap	The label of the roi in the corresponding rois files	The volume of the segmented region in voxels	The length of the major axis of the region	Z-position of the centroid of the region	Y-position of the centroid of the region	X-position of the centroid of the region	Z-position of the center of mass of the region	Y-position of the center of mass of the region	X-position of the center of mass of the region	The diameter of a circle with the same area as the region	Max intensity in the region	Mean intensity in the region	Min intensity in the region	The ratio of the area of a sphere with the same volume as the region to the actual surface area of the region	Ratio of pixels in the region to pixels of the convex hull image	Volume of the region in physical units	Units of the volume	Fraction of the two domain that is overlapping	Distance between the centers of mass of the two domains in the x dimension	Distance between the centers of mass of the two domains in the y dimension	Distance between the centers of mass of the two domains in the z dimension	Distance between the centers of mass of the two domains in 3D	Units of the distances	Name of the ROI
Processed Data Column Link To Assay File	Image Name

Experiment Number	5
Comment[IDR Experiment Name]	idr0156-szabo-tadcnd/experimentE
Experiment Data DOI	https://doi.org/10.17867/10000206e
Experiment Sample Type	cell
Experiment Description	Using the same cells as in Mouse_ESC_TADs, immobilized cells on a coverslip were fixed and nuclei were counterstained with DAPI for CND segmentation without the FISH procedure. DAPI stained nuclei were then observed with Structured Illumination Microscopy (3D-SIM).
Experiment Size 5D Images: 	Average Image Dimension (XYZCT): 512x512x1x1x1	Total Tb: 0
Experiment Example Images
Experiment Imaging Method	structured illumination microscopy (SIM)
Experiment Imaging Method Term Source REF	Fbbi
Experiment Imaging Method Term Accession	FBbi_00000332
Experiment Organism	Mus musculus
Experiment Organism Term Source REF	NCBITaxon
Experiment Organism Term Accession	10090
Experiment Comments	3D-SIM.  3 independent nuclei were monitored in this experiment.

# assay files
Experiment Assay File	idr0156-experimentE-annotation.csv
Experiment Assay File Format	tab-delimited text
Assay Experimental Conditions	Mouse ESCs (E14Tg2a.4 cell line, MMRRC, UC Davis) were cultured in the same condition as in Experiment Mouse_ESC_TADs.
Assay Experimental Conditions Term Source REF
Assay Experimental Conditions Term Accession
Quality Control Description	Standard Mouse E14 cell line (E14Tg2a.4) from MMRRC, UC Davis were used. Same cells as in Experiment Mouse_ESC_TADs.

# Protocols
Protocol Name	Mouse ESCs standard culture	No treatment	DAPI staining without FISH	Otsu's method and watershed segmentation
Protocol Type	Cell culture	No treatment	DAPI staining after fixation	Image segmentation
Protocol Type Term Source REF	EFO	EFO	EFO
Protocol Type Term Accession	EFO_0003789	EFO_0003969	EFO_0011016
Protocol Description	Standard E14 cell line (E14Tg2a.4) from MMRRC, UC Davis, were cultured in the same condition as in Experiment Mouse_ESC_TADs. Two days before the labeling, approximately 150.000 cells were plated in a 6-well plate containing an autoclaved glass coverslip (170 ± 5 μm (ZEISS)) coated with 0.1% gelatin, with fresh medium replacement after one day. Mouse ESCs were grown on the coverslip for a total of 2 days and washed once with PBS before fixation.	No specific treatment	Cells were fixed, permeabilized for 10 min in PBS/0.5% Triton X-100, rinsed in PBS, and incubated for 10 min in PBS  with DAPI (final concentration at 1 μg ml−1) and washed again at least 3 times for 5 min each in PBS. Coverslips containing cells were mounted on slides with VECTASHIELD and sealed with nail polish. 3D-SIM imaging was performed with a DeltaVision OMX V4 microscope equipped with a x100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (Evolve 512B; Photometrics) camera for a pixel size of 80 nm. Diode lasers at 405, 488, 561 and 647 nm were used with the standard corresponding emission filters. Z-stacks were acquired by scanning the sample in the axial direction (z-step of 125 nm) using 5 phases and 3 angles per image plane. Raw images were reconstructed using SoftWorx v.6.5 (GE Healthcare Systems) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). The quality of reconstructed images was assessed using the SIMcheck plugin (doi: 10.1038/srep15915) of ImageJ v.1.52i.	A single z-slice was extracted and DAPI was segmented using Otsu’s method; objects smaller than 0.0144 μm2 were discarded. Watershed was applied to the complement of the scaled intensity values (0 to 1, without Gaussian filter) within ROIs defined by the DAPI segmentation.

# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession

# Feature Level Data Files (give individual file details unless there is one file per well)
Feature Level Data File Name
Feature Level Data File Format
Feature Level Data File Description
Feature Level Data Column Name
Feature Level Data Column Description

#  Processed Data Files
Processed Data File Name	idr0156-experimentE-processed.csv
Processed Data File Format	tab-delimited text
Processed Data File Description	Extrapolated diameters of CNDs measured with DAPI staining.
Processed Data Column Name	Image Name	average_CND_diameter
Processed Data Column Type	linking column	data
Processed Data Column Annotation Level	Image	image
Processed Data Column Description	Name of the image file	Extrapolated diameters of CNDs measured with DAPI staining
Processed Data Column Link To Assay File	Image Name

Experiment Number	6
Comment[IDR Experiment Name]	idr0156-szabo-tadcnd/experimentF
Experiment Data DOI	https://doi.org/10.17867/10000206f
Experiment Sample Type	cell
Experiment Description	Using staged 15-16 Drosophila embryos (Drosophila melanogaster), derived cells were immobilized on a coverslip, fixed and  nuclei were counterstained with DAPI for CND segmentation without the FISH procedure. DAPI stained nuclei were then observed with Structured Illumination Microscopy (3D-SIM).
Experiment Size 5D Images: 	Average Image Dimension (XYZCT): 150x150x1x1x1	Total Tb: 0
Experiment Example Images
Experiment Imaging Method	structured illumination microscopy (SIM)
Experiment Imaging Method Term Source REF	Fbbi
Experiment Imaging Method Term Accession	FBbi_00000332
Experiment Organism	Drosophila melanogaster
Experiment Organism Term Source REF	NCBITaxon
Experiment Organism Term Accession	7227
Experiment Comments	3D-SIM.  20 independent nuclei were monitored in this experiment.

# assay files
Experiment Assay File	idr0156-experimentF-annotation.csv
Experiment Assay File Format	tab-delimited text
Assay Experimental Conditions	Staged 15-16 Drosophila male embryos were collected from adult flies and derived cells were immoblized on a coverslip for the experiment.
Assay Experimental Conditions Term Source REF
Assay Experimental Conditions Term Accession
Quality Control Description	The adult phenotype of the Y-GFP reporter line (y[1], w[67c23];Dp(1;Y), y[+] P{ry+11} P{w[+mC]=ActGFP}JMR1) was verified before the experiment.

# Protocols
Protocol Name	Drosophila culture	No treatment	DAPI staining without FISH	Otsu's method and watershed segmentation
Protocol Type	fly egg laying and staged embryos	No treatment	DAPI staining after fixation	Image segmentation
Protocol Type Term Source REF	EFO	EFO	EFO
Protocol Type Term Accession	EFO_0003789	EFO_0003969	EFO_0011016
Protocol Description	Flies (Drosophila melanogaster) were raised in standard cornmeal yeast extract medium at 21°C. Embryos were collected on agar/vinegar plates at stages 15-16 after egg laying, equivalent to a development of 12-16 h at 25°C. Embryos were manually dechorionated on a double-sided adhesive tape and displayed on an agar/vinegar plate to avoid drying during manual selection under a GFP binocular. A Y-GFP reporter line was used to select WT GFP+ male embryos (Szabo et al., doi: 10.1126/sciadv.aar8082).	4-5 selected dechorionated male embryos were disposed in the center of a glass coverslip (170 ± 5 μm (ZEISS)) coated with 1:10 poly-L-lysine (catalog no. P8920; Sigma-Aldrich) and squeezed directly on the coverslip with a Dumont #55 tweezer to produce a monolayer of cells.	Cells were fixed, permeabilized for 10 min in PBS/0.5% Triton X-100, rinsed in PBS, and incubated for 10 min in PBS  with DAPI (final concentration at 1 μg ml−1) and washed again at least 3 times for 5 min each in PBS. Coverslips containing cells were mounted on slides with VECTASHIELD and sealed with nail polish. 3D-SIM imaging was performed with a DeltaVision OMX V4 microscope equipped with a x100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (Evolve 512B; Photometrics) camera for a pixel size of 80 nm. Diode lasers at 405, 488, 561 and 647 nm were used with the standard corresponding emission filters. Z-stacks were acquired by scanning the sample in the axial direction (z-step of 125 nm) using 5 phases and 3 angles per image plane. Raw images were reconstructed using SoftWorx v.6.5 (GE Healthcare Systems) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). The quality of reconstructed images was assessed using the SIMcheck plugin (doi: 10.1038/srep15915) of ImageJ v.1.52i.	A single z-slice was extracted and DAPI was segmented using Otsu’s method; objects smaller than 0.0144 μm2 were discarded. Watershed was applied to the complement of the scaled intensity values (0 to 1, without Gaussian filter) within ROIs defined by the DAPI segmentation.

# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession

# Feature Level Data Files (give individual file details unless there is one file per well)
Feature Level Data File Name
Feature Level Data File Format
Feature Level Data File Description
Feature Level Data Column Name
Feature Level Data Column Description

#  Processed Data Files
Processed Data File Name	idr0156-experimentF-processed.csv
Processed Data File Format	tab-delimited text
Processed Data File Description	Extrapolated diameters of CNDs measured with DAPI staining.
Processed Data Column Name	Image Name	average_CND_diameter
Processed Data Column Type	linking column	data
Processed Data Column Annotation Level	Image	image
Processed Data Column Description	Name of the image file	Extrapolated diameters of CNDs measured with DAPI staining
Processed Data Column Link To Assay File	Image Name

Experiment Number	7
Comment[IDR Experiment Name]	idr0156-szabo-tadcnd/experimentG
Experiment Data DOI	https://doi.org/10.17867/10000206g
Experiment Sample Type	cell
Experiment Description	Using staged 15-16 Drosophila embryos (Drosophila melanogaster), derived cells were immobilized on a coverslip, fixed and submitted to the DNA FISH procedure using an Oligopaint probe targetting a drosophila TAD of 110 kb located on the X Chromosome. Coverslips with hybridized cells were then observed with Structured Illumination Microscopy (3D-SIM). This approach allows for the description of TAD organization in Drosphila cells. In this species, TADs ressemble CNDs in term of genomic size, diameter and volume.
Experiment Size 5D Images: 	Average Image Dimension (XYZCT): 55x55x56x1x1	Total Tb: 0
Experiment Example Images
Experiment Imaging Method	structured illumination microscopy (SIM)
Experiment Imaging Method Term Source REF	Fbbi
Experiment Imaging Method Term Accession	FBbi_00000332
Experiment Organism	Drosophila melanogaster
Experiment Organism Term Source REF	NCBITaxon
Experiment Organism Term Accession	7227
Experiment Comments	3D-SIM.  A probe of 110kb corresponding to a Drosophila TAD on Chromosome X was monitored in this experiment.

# assay files
Experiment Assay File	idr0156-experimentG-annotation.csv
Experiment Assay File Format	tab-delimited text
Assay Experimental Conditions	Staged 15-16 Drosophila male embryos were collected from adult flies and derived cells were immoblized on a coverslip for the experiment.
Assay Experimental Conditions Term Source REF
Assay Experimental Conditions Term Accession
Quality Control Description	The adult phenotype of the Y-GFP reporter line (y[1], w[67c23];Dp(1;Y), y[+] P{ry+11} P{w[+mC]=ActGFP}JMR1) was verified before the experiment.

# Protocols
Protocol Name	Drosophila culture	No treatment	Oligopaint FISH	Otsu's method and watershed segmentation
Protocol Type	fly egg laying and staged embryos	No treatment	DNA FISH	Image segmentation
Protocol Type Term Source REF	EFO	EFO	EFO
Protocol Type Term Accession	EFO_0003789	EFO_0003969	EFO_0011016
Protocol Description	Flies (Drosophila melanogaster) were raised and embryos were processed as described in Experiment Drosophila_DAPI. To analyze the 110 kb Drosophila TAD in an haploid context, that is, on chromosome X in male embryos, a Y-GFP reporter line (see genotype above) was used to select WT GFP+ male embryos (Szabo et al., doi: 10.1126/sciadv.aar8082). For the sake of homogeneity and simplicity, the same Drosophila line was used in Experiment Drosophila_DAPI.	4-5 selected dechorionated male embryos were disposed in the center of a glass coverslip (170 ± 5 μm (ZEISS)) coated with 1:10 poly-L-lysine (catalog no. P8920; Sigma-Aldrich) and squeezed directly on the coverslip with a Dumont #55 tweezer to produce a monolayer of cells.	Cells were hybridized with the Oligopaint fluorescent probe using a DNA FISH protocol, which is briefly described in the article and a detailed protocol can be found in Szabo et al. (PMID: 32820407; DOI: 10.1007/978-1-0716-0664-3_13). Here, the Oligopaint probe was labeled with ATTO 565 fluorophore. The coordinates, the size and the number of oligos for the Drosophila TAD probe can be found in the Extended Data Fig.9 of the article. Each oligo of the probe carries 3 fluorophores. Nuclei were counterstained with DAPI. Coverslips containing cells were mounted on slides with VECTASHIELD and sealed with nail polish. 3D-SIM imaging was performed with a DeltaVision OMX V4 microscope equipped with a x100/1.4 numerical aperture (NA) Plan Super Apochromat oil immersion objective (Olympus) and electron-multiplying charge-coupled device (Evolve 512B; Photometrics) camera for a pixel size of 80 nm. Diode lasers at 405, 488, 561 and 647 nm were used with the standard corresponding emission filters. Z-stacks were acquired by scanning the sample in the axial direction (z-step of 125 nm) using 5 phases and 3 angles per image plane. Raw images were reconstructed using SoftWorx v.6.5 (GE Healthcare Systems) using channel-specific optical transfer functions (pixel size of reconstructed images = 40 nm). The quality of reconstructed images was assessed using the SIMcheck plugin (doi: 10.1038/srep15915) of ImageJ v.1.52i.	For comparison with DAPI  (Experiment F), a single z-slice was randomly selected between the minimum and maximum z coordinate of the FISH-segmented object. Watershed was then applied as for DAPI, within ROIs defined by the probe segmentation.

# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession

# Feature Level Data Files (give individual file details unless there is one file per well)
Feature Level Data File Name
Feature Level Data File Format
Feature Level Data File Description
Feature Level Data Column Name
Feature Level Data Column Description

#  Processed Data Files
Processed Data File Name	idr0156-experimentG-processed.csv
Processed Data File Format	tab-delimited text
Processed Data File Description	Diameters and count of the CNDs.
Processed Data Column Name	Image Name	CND_count	TAD_diameter
Processed Data Column Type	linking column	data	data
Processed Data Column Annotation Level	Image	image	image
Processed Data Column Description	Name of the image file	Number of CNDs within a TAD after watershed segmentation	Diameter of the TAD
Processed Data Column Link To Assay File	Image Name