(optional) Displaying cluster descriptors Timing 10 min The Get_histograms.m code provided allows the generation of user definable histograms of all six key cluster descriptors, saved as .fig files to facilitate visualisation and interpretation of the data. This step is purely aesthetic. Open Matlab. Click on the folder” icon. Select the parent folder (“Condition i” folder in our “current example) (Figure 6). The files contained in this folder, including the .m files, will appear in the current folder section (on the left of the Matlab interface). Double click on the Get_histograms.m file to open the function in the editor. Enter the folder name containing the postprocessed data on line 4 (ROIs in our example) Enter the bin width which will be used to generate the histograms of each descriptor (lines 7-12). Click Run. The code will generate formatted histograms of each descriptor. TROUBLESHOOTING Timing Selecting and formatting regions of interest: 60 mins (for 30 regions) Preparing analysis: 5 mins Analysis processing time 45 mins (per region) Post-processing and visualisation: 15 mins Troubleshooting Problem Possible reason Solution 1A.v Localisations within the Low transfection Optimise transfection protocol or live preview are very efficiency or expression of immunolabelling procedure to sparse or there are the fluorescent construct, produce higher signal and lower or significant background low labelling efficiency of lower background localisations outside of the antibody or high the cell levels of non-specific binding 1A. xii .csv data file does not Incorrect path was Export with correct path to exist in the parent folder entered when exporting the parent folder the .csv file 1A. When running Either Coord.txt or Make sure config.txt and Coord.txt xxxii formatting.R, the correct config.txt are missing are in the parent folder. files are not created in from the parent folder. Ensure the working directory has Or the folder structure been set correctly (Figure 4) 1B. xx
(optional) Displaying cluster descriptors Timing 10 min The Get_histograms.m code provided allows the generation of user definable histograms of all six key cluster descriptors, saved as .fig files to facilitate visualisation and interpretation of the data. This step is purely aesthetic. Open Matlab. Click on the “current folder” icon. Select the parent folder (“Condition i” folder in our example) (Figure 6). The files contained in this folder, including the .m files, will appear in the current folder section (on the left of the Matlab interface). Double click on the Get_histograms.m file to open the function in the editor. Enter the folder name containing the postprocessed data on line 4 (ROIs in our example) Enter the bin width which will be used to generate the histograms of each descriptor (lines 7-12). Click Run. The code will generate formatted histograms of each descriptor. TROUBLESHOOTING Timing Selecting and formatting regions of interest: 60 mins (for 30 regions) Preparing analysis: 5 mins Analysis processing time 45 mins (per region) Post-processing and visualisation: 15 mins Troubleshooting Problem Possible reason Solution 1A.v Localisations within the live preview are very sparse or there are significant background localisations outside of the cell Low transfection efficiency or expression of the fluorescent construct, low labelling efficiency of the antibody or high levels of non-specific binding Optimise transfection protocol or immunolabelling procedure to produce higher signal and lower or lower background 1A. xii .csv data file does not exist in the parent folder Incorrect path was entered when exporting the .csv file Export with correct path to the parent folder 1A. xxxii Or 1B. xx When running formatting.R, the correct files are not created in the folder structure (Figure 4) Either Coord.txt or config.txt are missing from the parent folder. Make sure config.txt and Coord.txt are in the parent folder. Ensure the working directory has been set correctly
Revised_Nature_Protocols_FINAL.docx