README: Dynamic 1D Search and Processive Nucleosome Translocations by RSC and ISW2 Chromatin Remodelers'a0

Raw data of single molecule diffusion on naked DNA or on nucleosome arrays was collected on DNA stretched using dual optical tweezers and imaged for fluorescence using point scanning confocal microscopy. Initial raw data is in the form of an h5 file, which contains correlated metadata. However, due to the very large initial file sizes, we have chosen to upload images of kymographs (as tiff files) used for single particle tracking along with the respective single particle trajectories for both the red and green channel (as csv files). Frame-rates and pixel sizes are the same for all kymographs (0.0424 s and 0.1 um respectively). Single particle tracking was performed using LUMICKS Pylake Kymotracker as described in the methods section of our associated manuscript.

The uploaded data can be found in three parts as zip files:
(1) naked DNA data: remodeler diffusion on naked lambda DNA
(2) nucleosome array data: remodeler diffusion on nucleosome arrays
(3) remodeler-remodeler data: diffusion of remodelers labeled in red and green diffusing on the same naked lambda DNA

The csv files uploaded contain the following data as comma delimited columns (all in column 1 of the csv file):

(1)Line index (this is the trajectory number)
(2)Time (pixels): to convert to seconds 1 pixel = 0.0424 s
(3)Coordinate (pixels): to convert to distance 1 pixel = 0.1 um
(4)Time (s)
(5)Position (um)
(6)Counts (summed over 7 pixels): intensity of the molecule

Tiff files can be used as they are or can be converted to jpeg using software such as FIJI

Folders/Subfolders

Main Folders: "naked DNA", "nucleosome array", "remodeler-remodeler".

Each main folder name describes the experimental conditions that help identify the contained data with the section of the manuscript where the data is used. For example: all diffusion data of single remodelers collected on naked DNA is in the "naked DNA folder". For single remodelers on the nucleosome array, data is in the "nucleosome array" folder. For data of two remodelers on the same piece of naked DNA, the data is in the folder "remodeler-remodeler".

Subfolders: Given the description of the contents of the main folders, the user will be able to understand which data is contained in the subfolders by the description included in the name of the subfolder. For example, in the "naked DNA" main folder, the subfolder "ISW2_1mMATP" includes data of ISW2 diffusion in the presence of 1mM ATP. In the case of the "nucleosome array" main folder, the subfolder "ISW2" with subfolder "ATP" includes data on ISW2 diffusion in the presence of ATP on the nucleosome array. In the case of the "remodeler-remodeler" main folder, the subfolder "RSC-ISW2" includes data of where RSC and ISW2 are labeled in different colors and are diffusing on the same piece of DNA.

Code/Software

Source codes are written in Matlab
Analysis of the resulting single particle trajectories (csv files) can be performed as follows, using the provided Matlab source code. The result of this analysis are parameter files for each single particle trajectory detected within each kymograph. These parameter files contain amongst other parameters the generalized diffusion coefficient. Further processing of these files produces summary files for each condition: '93DValuesList.csv'94: mean diffusion coefficient values per diffusive class [i.e. non-diffusive, low-diffusion, high-diffusion], '93DwellTimeList.csv'94: list of dwell-times in a diffusive state after transitioning into that diffusive class, '93Parameter_Summary.csv'94: relevant parameters used for the analysis [i.e. frame-rate, rolling-window size, threshold values differentiating diffusive classes], and '93StatePercentagesPerTrajectory.csv'94: percentage of time spent in a given diffusive class per trajectory.

Run '93c01_smoothKymo.m'94 and select the folder containing the csv files (this needs to be done twice in the case of two-color channels, once for the data in the red channel and once for the data in the green channel). This step smooths the tracking data, which will be the input for subsequent analysis.

2.'a0'a0'a0'a0 Run the rolling-window analysis.'a0 This must be run once for data in the red channel and a second time for data in the green channel.'a0 Rather than selecting the folder of interest as was done in step 1, this step was designed to feed in all data folders for a given remodeler. Select the folder containing all data folders. These sub-data folders must have '93_csvfiles'94 at the end of the folder name to be detected for batch processing.

a.'a0'a0'a0'a0 For data in the red color channel: run '93c02_1_noPlot_Rollingwindow_CalculateParameters_Plot_AllCSV_VER2.m'94

b.'a0'a0'a0'a0 For data in the green color channel: run '93c02_2_ALT_noPlot_Rollingwindow_Nucleosomes.m'94

3.'a0'a0'a0'a0 Finally, in the same was a was done in step 2, run: '93c03noplotCompute_DStates_fromcsv_NakedDNA_stringentsliceminimum.m'94 to create summary information for the condition of interest.

Functions used in this analysis are provided in the source code folder. When running the scripts, please select change folder so that the functions can be found. Note: @msdanalyzer was used for calculating diffusion coefficients. This Matlab package needs to be downloaded and added to the same folder as the functions provided in order to run.

Nadine Tarantino, Jean-Yves Tinevez, Elizabeth Faris Crowell, Bertrand Boisson, Ricardo Henriques, Musa Mhlanga, Fabrice Agou, Alain Isra'ebl, and Emmanuel Laplantine. TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO-IKK supramolecular structures. J Cell Biol (2014) vol. 204 (2) pp. 231-45

An example of the complete analysis is provided for the following data set: nucleosome array -> ISW2 -> ATP -> 20211117_example_csvfiles

The resulting analysis is added as subfolders of the respective color channels, with each subsequent analysis creating a new subfolder.