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Detailed Instructions

Create a configuration file

  1. Create a configuration file
    • To create a new Configuration file, open RainFlow and go to File > New > New Document from the menu bar. Then navigate through the steps in the sidebar.
  2. Laser
    • Add the names and/or wavelengths of the laser lines used in your experiment. You can add them manually using the + button.
  3. Channels
    • Here you can add information about the detection channels used in your experiment.
    • RainFlow can automatically extract metadata from a sample FCS file to record the names and voltages for each detection channel.
    • RainFlow will also record the timestamps present in the Time channel and denote it with a clock icon. If the Time channel is not automatically detected, you can select the correct channel and click on the Set button that will appear next to it, so set it as the Time channel.
    • Next, assign each channel to one of Scatter or Fluorescence depending on the type of light collected.
    • Assign each Fluorescence detection channel to the correct excitation laser line by choosing from the list of lasers specified in the Laser tab.
  4. Bead Detection
    • Beads refer to Rainbow calibration particles. To set up bead detection parameters, you will need to:
      • Select the Scatter channels to use for this step which measure the Forward Scatter Area, Forward Scatter Height, and Side Scatter Height.
      • Select the expected number of bead peaks, which depends on the type of Rainbow beads used in your runs. You can choose from a range of 3-8 peaks which is the range for most commercially available Rainbow beads.
      • Select channels for bead detection. Depending on your experimental configuration, all bead peaks may not be fully resolved in the linear range of detection for every detection channel. For RainFlow to correctly identify beads from cell events, select only the Fluorescence channels in which all expected bead peaks are fully resolved.
  5. Once the Configuration File is ready, the sidebar will change to show all navigation bar items’ status a green dot, and you can save the configuration file for repeated use.

 

Create a Workspace file

  1. Create a Workspace file
    • Once you have a configuration file saved, you can open it and use the Create Workspace button on the top right corner to create a new workspace file.
    • When you open a new workspace file, the navigation sidebar will show you all the analysis steps to go through. The colored dot next to each step denotes the status – grey refers to step not started, orange refers to step incomplete, and green refers to step completed successfully.
  2. Configuration
    • This tab links to the RainFlow configuration file associated with this workspace. You can confirm or replace the linked configuration file from this tab.
  3. Samples
    • Use this tab to import the sample FCS files for analysis using the Import FCS Files button.
    • Once files are imported, RainFlow will extract the metadata from each FCS file and make sure that sample collection parameters match the parameters specified in the linked configuration file. Is the parameters match, the sample is valid and denoted with a green dot.
    • At the top, you will see three additional options for each sample
      • General – displays sample metadata
      • Timelines – displays time course for events detected across different channels
      • Histograms – displays event intensity histograms for all channels
  4. Detect Beads
    • In this step, the user selects the samples for which beads will be detected. Once selected, click on the Detect Beads… button, and then on the Start button in the pop-up window.
    • Once bead detection is complete, click on Show detected beads histograms, or on the Histograms tab at the top, to view sample-wise bead histograms across channels.
      • Here you will see information such as the total number of bead events detected, the bead count for each expected bead peak, as well as the Gaussian Mixture Model parameters (mean and variance) for each bead peak.
      • RainFlow also calculates a quality score for each channel and sample based on the detected bead properties.
  5. Quality Validation
    • The sample by channel quality scores calculated in the previous step are displayed as a color-coded table to aid users in selecting the samples that are high-quality enough to be taken forward for analysis.
    • Users first select the Minimum Quality Score threshold. This can either be a default recommended value of 15. Alternatively, users can also set a custom threshold score.
    • Then users can use the Select high quality samples button to automatically select all samples that pass the minimum quality threshold.
    • Once high-quality samples are selected, click the Validate button to validate the workspace and lock it from further changes. You will see a lock icon in the sidebar once the workspace is validated.
  6. Compensation
    • The validated workspace samples can now be compensated by importing a spillover matrix using the Import CSV file button, and clicking on Compensate.
    • Users can check the data histograms before and after compensation by toggling the Source and Compensated toggle buttons under Histograms.
    • Users can also choose to see histograms for the Full data, Only beads, or Only possible cells.
  7. Alignment
    • Next, choose a reference sample from one of the validated samples. This will be used as the baseline for aligning all other sample bead peaks.
    • Once aligned, users can check the data histograms before and after alignment by toggling the Source, Compensated or Aligned toggle buttons under Histograms.
    • An arrow head will mark the reference sample used for alignment.
  8. Final Report
    • Once the data are validated, compensated and aligned, users can export the analyzed data and metadata using the Export.. button at the top left corner.
    • Select an output filename and click Export in the pop-up window that shows up. This will export a ZIP file which will contain the following csv files for each sample:
      • eventTypes: Each event is coded as follows:
        • 0 – non-bead events. Could be cells, debris, etc.
        • 1 – potential bead event as detected by scatter properties, but not selected in the bead-refinement step.
        • 100 + n – validated bead event, where n is the peak number starting from 0, such that peak0 (lowest intensity bead peak) are denoted as 100, peak1 as 101, and so on.
      • compensatedData: compensated data before alignment to reference
      • alignedData: data aligned to chosen reference sample after compensation
      • beadsDetectedData: bead peak parameters such as channel mean and variance for each peak as estimated by RainFlow
    • A metadata.json file is also exported for the workspace, which contains the file original name and ID name used for export for each sample file, as well as associated metadata relevant to the analysis.
  9. Save your workspace
    • You can save your workspace file at any time during or after the analysis.