Description of deposited data
This repository contains data and analysis code for the manuscript:
"Spatiotemporal kinetics of the SRP pathway in live E. coli cells"	
Ivan L Volkov, Erik Lundin, Kalle Kipper, Mikhail Metelev, Spartak Zikrin, and Magnus Johansson*	
Department of Cell and Molecular Biology, Uppsala University	
*Correspondence: m.johansson@icm.uu.se	

DOI: 10.17044/scilifelab.20502126

Update date: September 8, 2022


--List of archives--
The data have been separated into several zip archives according to:

LD655.zip			raw data and tracking of 4.5S RNA labeled with LD655 dye
Cy5.zip				raw data and tracking of 4.5S RNA labeled with Cy5 dye
HMM_analysis.zip		HMM analysis of tracking data of 4.5S RNA labeled with LD655 dye and Cy5 dye
Growth_data.zip			cell growth data used in Fig. S3 and S4
Simulations.zip			scripts and data related to microscopy simulation
Scripts_for_figures.zip		scripts used to generate figures for the manuscript
Tools.zip			software code and scripts used for data analysis
pQE30Mod-lacY-Halotag.zip	annotated DNA sequence of the pQE30Mod-lacY-Halotag plasmid in .dna format

"LD655" and "Cy5_manuscript" folders contain:
- data of individual microscopy experiments (e.g. "2018_07_13_45SRNA-****")
- "Parameter" folder with parameter file used for segmentation, dot detection and tracking
- "EMCCDcalibration_offset_20ms_gain104.tif" file with camera offset used in dot detection
- "runTrackingArray.m" and "jobArray.sh" to run job on a server			

Folders with individual microscopy experiments have the following content:                   

- "RawData" 
Original (raw) microscopy images. Within subfolders for each position (PosXX), corresponding to each imaged bacterial mini-colony, there are three phase contrast images taken with different Z offset (in folder A_Phase), a bright field image (B_Bf), fluorescent movies (C_638_20ms) and fluorescent Sytox images (D_405).
- "Output_uWin2_uMinTrack3***" 
Preprocessed phase contrast images, cell segmentation masks and dot detection/tracking output data.
- "Output_TransMat" 
Transformation matrix generated to transform phase contrast (cell segmentation) coordinates to fluorescence and bright field coordinates (required due to acquisition of data on different cameras).
- "SoftlinkedDataForAnalysis" Soft links to the original raw microscopy images (from "RawData" folder) that are sorted by image positions. Used by the image analysis pipeline.
- "SoftlinkedDataToLookAt" 
Soft links to the original raw microscopy images (from "RawData" folder), segmented images, and fluorescent images used for analysis that are sorted based on the type of the images to facilitate visual examination of images. 


"HMM_analysis_Cy5" and "HMM_analysis_LD655" folders contain:
- tracking data of corresponding tracking experiments (e.g. "2018_07_13_45SRNA-****")
- HMM results in "HMMresults_uWin2_uMinTrack3_Tmin5" folder
- HMM Parameter file "uWin2_uMinTrack3_Tmin5.m"
- "trjExport.m" script for HMM analysis
- "runTrackingArray.m" and "jobArray.sh" to run job on a server


--Tools--
Contains the Image Analysis Pipeline, HMM pipeline for processing of images obtained in microscopy experiments, plus additional analysis scripts. Read "README.txt" file for instructions.
"HMMpipeline_2019_12_10_Spartak_coarse-graining" HMM pipeline was used for analysis of most of the data.
"HMMpipeline_2021_02_08" has the same functionality plus allows states with zero occupancies after coarse-graining. Used to coarse-grain HMM results from experiments with FtsY depletion and 4.5S RNA overexpression. 

--Scripts_for_figures--
Code to generate figures presented in the manuscript and Dataset 1 table.


--Growth_after_electropor_EXP-21-BK4792--
Contains cell growth data used in Fig. S3 and S4.


--simulations--
Contains simulation models, data, downstream analysis and scripts of microscopy simulation. Read README.txt files in "simulations" and "simulations/only_scripts" folders for instructions.


--Licence--
CC BY 4.0

--Contact--
m.johansson@icm.uu.se