Uniform dynamics of cohesin-mediated loop extrusion in living human cells

Overview

This repository contains all the raw and quality-controlled distance times series associated with the study "Universal dynamics of cohesin-mediated loop extrusion", as well as the code to analyze them. Here, we provide the following information:

• The cell lines and conditions used in this study
• A summary of how the data was collected and processed
• The structure of the distance time series

Cell lines and conditions

In total, the dataset covers 14 experimental conditions with 5 different cell lines, 2 different treatments, and 2 different imaging acquisition frequencies, listed hereafter as (Cell line; Treatment; Frequency):

• L1; - Auxin
• L1; + Auxin 2h
• L2; - Auxin
• L2; + Auxin 2h
• T1; - Auxin
• T1; + Auxin 2h
• Half TAD; - Auxin
• Half TAD; + Auxin 2h
• Adjacent; - Auxin
• L2; - Auxin; High frequency
• L2; + Auxin 2h; High frequency
• Half TAD; - Auxin; High frequency
• Half TAD; + Auxin 2h; High frequency
• Adjacent; - Auxin; High frequency

Data and data processing

Time lapse image acquisition was performed with an inverted microscope (Nikon) coupled to the Dragonfly spinning disk (Andor) using a 100X Plan Apo 1.45 NA oil immersion objective. Excitation sources were 488 nm (150 mW) and 637 nm (140 mW) lasers. Exposure time was set to 85 ms for both channels with 1% laser power in far-red and 5-8% laser power in the GFP channel depending on the imaged cell line. Z-stacks of 29 optical slices separated by 0.29 µm each were acquired every 30 s (or every 9 s for 'HighFreq' datasets) using the perfect focus system and five different stage positions were imaged for each 2-hour (or 36 min for 'HighFreq' datasets) acquisition. The two channels were acquired simultaneously on two distinct EMCCD iXon888 cameras (1024 x 1024 pixels, effective pixel size: 0.121 µm).

The 3D image time series were processed as described at https://github.com/imodpasteur/Sabate_et_al_TAD_Anchors/tree/main/Live-cell_analysis/Image_processing to obtain time series of 3D distances from live-cell microscopy images of TAD anchors. Time series have been corrected for chromatic aberrations. The 'Code_for_quantification' file contains the python code used to analyze time series, also available at: https://github.com/imodpasteur/Sabate_et_al_TAD_Anchors (version v0).

Data are provided in two different formats: the unfiltered data and the quality-controlled (QC) data. The unfiltered data contains raw localizations of fluorescent spots from cells in G1. The quality-controlled data were generated from the unfiltered data after filtering of localization and tracking errors. This latter dataset was used for all analyses of the article. We also provide the datasets acquired at a higher imaging frequency, which contain 'HighFreq' in their names.

File names are formatted follows.

• Quality-controlled time series: {CellLine_Treatment_ImagingFrequency_}QC.csv
• Unfiltered time series: {CellLine_Treatment_ImagingFrequency_}Unfiltered.csv

Structure of Data

The trajectory data are provided as .csv files consisting of 19 columns. The column headers are:

• Track_pair: a unique index for a pair of fluorescent spots
• Track_1_id: trajectory index of spot 1
• Track_1_id.1: trajectory index of spot 2
• Frame: the frame at which the pair of spots was localized
• Spot_1_X: x coordinate of the spot in the far-red channel (units in µm)
• Spot_1_Y: y coordinate of the spot in the far-red channel (units in µm)
• Spot_1_Z: z coordinate of the spot in the far-red channel (units in µm)
• Spot_2_X: x coordinate of the spot in the green channel (units in µm)
• Spot_2_Y: y coordinate of the spot in the green channel (units in µm)
• Spot_2_Z: z coordinate of the spot in the green channel (units in µm)
• Distance: 3D distance between the two fluorescent spots (units in µm)
• Precision_1_X: localization precision of the spot in the far-red channel in the x coordinate (units in µm)
• Precision_1_Y: localization precision of the spot in the far-red channel in the y coordinate (units in µm)
• Precision_1_Z: localization precision of the spot in the far-red channel in the z coordinate (units in µm)
• Precision_2_X: localization precision of the spot in the green channel in the x coordinate (units in µm)
• Precision_2_Y: localization precision of the spot in the green channel in the y coordinate (units in µm)
• Precision_2_Z: localization precision of the spot in the green channel in the z coordinate (units in µm)
• precision_Distance: localization precision of the distance between the two spots (units in µm)
• Score: weight associated with each 'precision_Distance', used in all downstream analyzes.