Supplementary Data 1: Prepare data for cyCONDOR analysis

Step 1

Within the FlowJo workspace, you can adjust compensation as needed and optionally apply a basic gating hierarchy to your data. This is recommended to remove debris and unwanted cells, reducing the size of your final dataset and improving computational efficiency. For instance, during immune cell analysis, consider gating on CD45+ cells for cyCONDOR analysis.

Step 2

Right-click in the gate you want to export and select Export/Concatenate Populations

Tip: If you want to export this gate for all the samples in the workspace, before this step right-click on the desired gate and select Select Equivalent Nodes to select the same gate in all samples.

Exemplary FlowJo workspace (left) and exemplary option window shown after right-clicking on the desired gate

Step 3

Now, choose the format for your exported data. FCS3 is the recommended format. Additionally, select the destination folder where you want to save the file. You have two options for selecting which channels to export:

• Export all compensated values: This option is selected in the image below and will export all channels that have been compensated.
• Manually select compensated channels: This option allows you to choose specific compensated channels to include in the exported .fcs file.

Note: If data were not compensated in FlowJo, for example cyTOF data or BD S8 unmixed data, export uncompensated values.

Population Export or Concatenate window

Step 4

Click on Export. Now all selected files will be exported in the selected folder. As input for cyCONDOR the data_path should only contain the .fcs files intended for the analysis.

Exemplary folder with exported .fcs files

Step 5

The annotation table, used to load .fcs files, should have the file names of all .fcs files in the first column. Additional columns can be included to store any relevant metadata for your analysis. There are no restrictions on the type of information you can include in these metadata columns. We advise to save the annotation table in a separate folder and save it as comma separated .csv file. An example of annotation table is shown below:

Exemplary metadata table visualized in Excel (left) and Notepad (right)