Below is the information on how samples were stained and images acquired, sorted by microscopy technique. Immunofluorescence staining Cells were grown to 80% confluency on 22x22 mm #1.5H high-precision coverslips (thickness 0.170 ± 0.005 mm, Marienfeld Superior). Cells were washed in PBS, pre-extracted in ice-cold PBS 0.2% Triton X-100 (Sigma-Aldrich) for 1.5 min on ice and fixed in 4% formaldehyde for 15 min. For non-pre-extracted samples, cells were fixed and subsequently permeabilized in PBS 0.2% Triton X-100 for 5 min. Primary and secondary antibodies were diluted in antibody diluent (DMEM medium containing 10% FBS, 0.05% sodium azide, sterile filtered 0.2 mm). Samples were incubated in primary antibody for 1.5 h and for 1 h in secondary antibody solution supplemented with 4',6'-diamidino-2-phenylindole-dyhydrochloride (DAPI, 0.5 mg/ml). In between primary and secondary antibody staining, samples were washed 3x in PBS 0.2% Tween. After secondary antibody incubation, samples were washed 3x again, samples were post-fixed in 4% formaldehyde for 15 min and samples were washed in PBS and distilled water. For QIBC, samples were mounted in Mowiol-based mounting medium (Mowiol 488 (Calbiochem)/glycerol/Tris-HCl, pH 8.5) and in non-hardening Slowfade Diamond (Thermo Fisher Scientific, S36963) for Spinning Disk and 3D-SIM imaging. For EdU staining, cells were incubated with 10 mM EdU 20 min before pre-extraction and EdU detection was performed before primary antibody incubation according to the manufacturer's instructions (Thermo Fisher Scientific). For detection of endogenously tagged proteins with chemical dyes, samples were incubated for 1.5 h with Halo- or 3 h with SNAP-ligands, washed three times in fresh media, and incubated for 30 min in the incubator in fresh media for exit wash of unbound dye. For experiments with two Halo dyes, cells were incubated with both dyes for 1.5 h, then washed and chased with a third dye as indicated. For calibration purposes, U2OS cells were incubated with 100 nm 4-colour Microspheres (fluorescent blue, green, orange, dark red; Thermo Fisher Scientific) for 3 days at a density of 30 ml bead slurry / 2x105 cells. For G2 analysis, cells with highest mean intensities for Cyclin A (fixed cells) or sororin (pre-extracted cells) were chosen. DNA FISH Methanol-acetic acid–fixed cells were prepared and hybridized as described in (Brown et al., 2006). Hybridized slides were examined using a fluorescence microscope (Olympus BX60). Hybridization efficiency was assessed by scoring FITC signals in ten metaphase spreads and only those hybridizations with greater than 80% efficiency were scored. 200 FITC signals were scored as either single or split dots for each hybridization. The percentage of split dot values for each cosmid plotted represent the average of two independent biological experiments. RASER-FISH RASER-FISH allows DNA sequence detection in intact 3D cell nuclei as it maintains fine-scale chromatin structure by replacement of heat denaturation with exonuclease III digestion after UV-generation of DNA nicks. RASER-FISH was carried out as described in (Brown et al., 2022; Ochs et al., 2019). In brief, U2OS cells were seeded on 22x22 mm #1.5H high-precision coverslips (thickness 0.170 ± 0.005 mm) and labelled for 18 h with 10 mM BrdU/BrdC mix (3:1). Cells were either pre-extracted and fixed as described above or fixed in 4% formaldehyde and permeabilized in PBS 0.2% Triton-X-100 for 5 min. Immunofluorescence staining for sororin or Cyclin A was carried out as described above. After incubation with 0.5 mg/ml DAPI for 15 min for UV sensitization, cells were treated with UV light (254 nm) for 15 min followed by incubation with 5 U/ml exonuclease III (NEB) at 37°C for 15 min. Biotin labelled probes were denatured in hybridization mix at 90°C for 10 min, pre-annealed with human Cot-1 DNA (Invitrogen) at 37°C for 15 min and hybridized to samples overnight at 39°C. Coverslips were washed twice in 1x SSC buffer at 37°C for 30 min, once in 1x SSC at room temperature, once in PBS, and biotin was detected by incubation with streptavidin-Alexa488 (1:500, Thermo Fisher). Samples were post-fixed, rinsed in PBS and MilliQ and mounted in Slowfade Diamond. For RAD21 degradation, cells harboring RAD21-Halo were incubated with 2.5 mM HaloPROTAC3 (Promega) for the last 8 h of BrdU/BrdC incubation. For dye mixing experiments, dye mixing was carried out as described above for the last 2 h of BrdU/BrdC incubation. The probes for all FISH assays in this study were obtained from a chromosome 16p specific cosmid library based on genome assembly NCBI36 (Stallings et al., 1990) and have been characterized in (Daniels et al., 2001) for mapping of the terminal 2 Mb of chromosome 16 p-arm. The GenBank accession number for the entire map is AE005175 and the specific probes used in this study have EMBL IDs HS306A4 (AL008727) and HS443D9 (Z92845). Probes were biotin-labelled. Quantitative image-based cytometry (QIBC) QIBC (Ochs et al., 2016; Toledo et al., 2013) was performed on a motorized Olympus IX83 inverted wide-field microscope equipped with Semrock DAPI/FITC/Cy3/Cy5 Quad LED filter set, Hamamatsu Orca Fusion B CMOS camera and Lumencor SPECTRA X light source. Automated unbiased image acquisition was carried out with the proprietary ScanR acquisition software. Identical exposure times were used for all samples within one experiment and settings were chosen for maximum dynamic range under non-saturating conditions. Most data was obtained with the Olympus UPLXAO 20x, NA 0.8 air objective. Depending on cell confluency and type of image analysis, 49-100 images were acquired, aiming for at least 2000 cells per analysis after gating. After acquisition, images were analyzed using the ScanR analysis software. DAPI signal was used for segmentation of nuclei based on intensity threshold. This nucleus mask was then used to quantify pixel intensities in the different channel for each individual nucleus. After segmentation and pixel quantification, measured parameters were extracted (mean and total intensities, area, circularity, well). For automated vermicelli quantification, edge detection was used, and 4 arbitrary classes were defined based on increasing edges (none, mild, moderate, and severe chromatin compaction). All data were exported to Tibco Spotfire, which was used to quantify average/median values in cell populations and to generate color-coded scatter plots in a flow-cytometry fashion, or to GraphPad Prism to generate stacked bar charts. Spinning disk microscopy Representative pseudo-confocal images were acquired with an UltraView Vox spinning-disk microscope (Perkin Elmer) and Volocity software (version 6.3.1) with a 60x, 1.42 NA Plan-Apochromat oil-immersion objective. Images were captured with a Hamamatsu EMCCD 16-bit camera at a spatial resolution of 121x250 nm. Single z-slices of whole nuclei are shown, brightness and contrast were linearly adjusted for optimal display. Color-channels were false-colored. Structured illumination microscopy (SIM) 3D-SIM images were acquired with a DeltaVision OMX SR system (GE Healthcare) equipped with a 60x, 1.5 NA UPLAPO60XOHR oil immersion objective (Olympus), pco.edge 4.2 sCMOs cameras (PCO), and 405 nm, 488 nm, 568 nm and 640 nm lasers. 3D image stacks were acquired over the whole nuclear volume in z with 15 raw images per plane (3 angles, 5 phases). Spherical aberration was minimized using immersion oil with refractive index 1.514 and an objective collar setting of 0.140 for image acquisition. Raw data was computationally reconstructed with SoftWoRx 7.2.0 (GE Healthcare) using channel-specific optical transfer functions (OTFs) recorded using immersion oil with RI 1.516 and Wiener filter setting 0.0030. All SIM data were routinely and meticulously quality-controlled for effective resolution and absence of artifacts using SIMcheck (Ball et al., 2015). Multichannel acquisitions were aligned in 3D with Chromagnon software (Matsuda et al., 2020) using 3D-SIM acquisitions of multicolor EdU-labelled C127 cells as colocalization reference. Images were thresholded based on the MCNR function of SIMcheck, which generates a metric of local stripe modulation contrast in different regions of the raw data and directly correlates this with the level of high-frequency information content in the reconstructed data. Only immunofluorescent signals with underlying MCNR values that exceeded a stringent quality threshold were considered for further analysis, while localizations with low underlying MCNR values were discarded. Thereby, any SIM signal, which falls below reconstruction confidence and is considered to be a labelling/imaging artifact, is excluded from further data interpretation (Ball et al., 2015). For representative images, single z-slices, cropped regions of whole nuclei or partial z-stacks are shown as indicated in figure legends. Brightness was linearly adjusted for optimal presentation. Channels were false-colored. HILO photobleaching Cells were seeded and labelled on 35 mm diameter, No. 1.5 MatTek dishes, pre-extracted and fixed as described above, and imaged in PBS. For photobleaching, a custom-built total internal reflection fluorescence (TIRF) microscope (described in detail in (Szczurek et al., 2023)) was used with a 100x 1.4 NA oil objective (Olympus) and with an iChrome MLE MultiLaser engine (Topica Photonics). Photobleaching was performed using highly inclined laminar optical sheet illumination (HILO). Emission was projected onto the central region of an iXon 897 EMCCD camera (Andor, 512x512 pixels). Pixel size in acquired images was 96 nm. Sample position and focus were controlled with a motorized stage and z-motor (ASI). For photobleaching samples were exposed to 30 % (RAD21-Halo) and 10-20% (sororin-SNAP) 561 nm laser and imaged using 100 ms camera interval for up to 1.5 min. Obtained images were analyzed with the Fiji plugin "Time Series Analyser" (https://imagej.nih.gov/ij/plugins/time-series.html) and background subtraction over time was performed using parallel background measurements.

Funding provided by: Austrian Academy of SciencesCrossref Funder Registry ID: https://ror.org/03anc3s24Award Number: LS17-003 Funding provided by: Austrian Academy of SciencesCrossref Funder Registry ID: https://ror.org/03anc3s24Award Number: LS19-001 Funding provided by: Boehringer Ingelheim FondsCrossref Funder Registry ID: https://ror.org/00dkye506Award Number: PhD Fellowship Funding provided by: Cancer Research UKCrossref Funder Registry ID: https://ror.org/054225q67Award Number: 26747 Funding provided by: European Molecular Biology OrganizationCrossref Funder Registry ID: https://ror.org/04wfr2810Award Number: ALTF 1172-2019 Funding provided by: EPA Cephalosporin Found*Crossref Funder Registry ID: Award Number: CF361 Funding provided by: European Research CouncilCrossref Funder Registry ID: https://ror.org/0472cxd90Award Number: 101019039 Funding provided by: Danmarks Frie ForskningsfondCrossref Funder Registry ID: https://ror.org/05svhj534Award Number: 0164-00011B

Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin's stoichiometry in vivo. Using quantitative super-resolution imaging, we identify two discrete populations of chromatin-bound cohesin in post-replicative human cells. While most complexes appear dimeric, cohesin localized to sites of sister chromatid cohesion and associated with Sororin is exclusively monomeric. The monomeric stoichiometry of Sororin:cohesin complexes demonstrates that sister chromatid cohesion is conferred by individual cohesin rings, a key prediction of the proposal that cohesion arises from their co-entrapment of sister DNAs.