Scripts for Head-on and co-directional RNA polymerase collisions orchestrate bidirectional transcription termination

The python scripts used to extract data for analysis of Lumick's CTrap .h5 files are listed here. Short descriptions of what each script does are attached below. Additional details and suggested use cases/tutorials for each individual script are available on Lumicks' Harbor code-sharing repository (https://harbor.lumicks.com/).

CTrapVis.py ("CTrapVis" on Harbor)

This script is the second version of a GUI designed to dynamically look through .h5 files from a Lumicks C-Trap instrument and extract desired data for further analysis. This script allows users to take full advantage of the tools in the pylake library to visualize and extract data of interest without python scripting knowledge or the need for a computer with blue lake software. Users are able to choose what plots they want(Trap Position vs. Time, Force vs. Distance, Force vs. Time, RGB image, or a combination of RGB and one of the force/trap position options) with options to customize the plot to add a title, scale RGB image values, shift the axis of all available axis. This script can analyze kymograph, scans/multiple scans, and force-distance curves contained within a .h5 file.

kymotracker_calling_script.py ("Batch Kymotracker Script" on Harbor)

This script is designed to process TDMS and .h5 kymograph files and perform batch processing of tracked line foci for downstream analysis. Lines are tracked using either of Lumicks' two-line tracking algorithms available when kymotracker was first released– more information can be found at Lumicks'Kymotracking Tutorial. This script automatically exports the lines tracked for each of the colors requested (time data in seconds, coordinate data in nm) as well as the kymotracking settings for each call of the algorithm in a .xlsx file. Metadata from the files are stored in a separate .csv file. There are built-in options to extract the photon counts around the foci (as determined by the line_width variable)and the distance between a chosen foci color and the other color's foci. All additional data is stored in the same .xlsx file.

area_photon_counter.py ("Area Photon Count Extractor" on Harbor)

This script is used to extract the sum of photon counts in a line scan from chosen areas of a kymograph in a .h5 file. This analysis method is useful when you want to measure raw intensity values of fluorescent molecules to quantify how many molecules are present without worrying about potential biases introduced by using line tracking methods.

Package Versions Tested In:

• Python 3.8.6
• NumPy 1.19.2
• lumicks.pylake 0.8.1
• Matplotlib 3.3.2
• pandas 1.1.2
• tifffile 2020.10.1
• nptdms 1.5.0