Overview This repository contains all the raw and processed trajectory data associated with “paper title”. In this ReadMe file we provide the following information: The cell lines and conditions used in this study A summary of how the data was collected The structure of the chromosome locus tracking data Cell lines and conditions In total, the dataset covers 12 experimental conditions representing the following cell lines and treatment conditions: C36 C65 C27 CTCF-AID (untreated) CTCF-AID (2 hours AID) CTCF-AID (4 hours AID) RAD21-AID (untreated) RAD21-AID (2 hours AID) RAD21-AID (4 hours AID) WAPL-AID (untreated) WAPL-AID (4 hours AID) WAPL-AID (6 hours AID) Data and data processing Trajectories were obtained from 3D timeseries of mouse embryonic stem cell colonies in the conditions listed above using a LSM900 Airyscan 2 Zeiss microscope. For each movie we recorded 365 frames of 49.69 µm x 49.69 µm (584 x 584 pixels, pixel size: 0.085 µm by 0.085 µm), separated by an interval of 20 seconds for a total of just over 2 hours. 3D images were composed of 30 z-stacks separated by 0.25 µm, for a total height of 7.25 µm. Imaging was performed in two colors allowing the tracking of two arrays of fluorophores on Chromosome 18 near the Fbn2 gene. In all conditions, the fluorophore arrays were separated by 515 kb (except the C27 clone where separation was 10 kb). The 3D image time series were processed using ConnectTheDots: https://github.com/ahansenlab/connect_the_dots to obtain paired trajectories of chromosome loci over time. The trajectories have been corrected for chromatic shifts and aberrations. Data are provided in an “unfiltered” format (meaning that individual dot localizations were not quality control filtered) , or a filtered format (the same data set, but having undergone quality control). The filtered (quality controlled) trajectory data was used for all the quantitative analyses in the article “”. File names are formatted follows. Quality controlled data have the structure: {Clone_and_condition_name}.tagged_set.tsv Unfiltered data have the structure: {Clone_and_condition_name}.unfiltered.tagged_set.tsv For example, for RAD21-AID tagged clone, for imaging performed after two hours of protein degradation, the quality-controlled file name is: RAD21_2_hr.tagged_set.tsv. Please note that for all no-treatment conditions, we used “0 hours” as the tag. Thus, the RAD21 (untreated) becomes RAD21_0_hr.tagged_set.tsv. Structure of Data The trajectory data are provided as tab-separated text files consisting of 10 columns. The column headers are: id: a unique dot pair index t: the frame in which the dots were localized x: x-coordinate of the dot in the EGFP channel (units in µm) y: y-coordinate of the dot in the EGFP channel (units in µm) z: z-coordinate of the dot in the EGFP channel (units in µm) x2: x-coordinate of the dot in the mScarlet channel (units in µm) y2: y-coordinate of the dot in the mScarlet channel (units in µm) z2: z-coordinate of the dot in the mScarlet channel (units in µm) dist: 3D distance between the dots across channels (units in µm) movie_index: an identifier used to link the dot pair back to the raw image timeseries.

MG, HBB, SGH contributed equally to this study.