Basic Guide to viSNE Analysis

Cytobank Guide to viSNE Analysis
The Premier Platform for Single Cell Analysis
Basic Guide to viSNE Analysis
This tutorial walks you through analysis of a 26 parameter cytometry dataset.
First Steps
Log into fluidigm.cytobank.org or any enterprise Cytobank.
Note: You can sign up for a 30 day free trial at fluidigm.cytobank.org to gain access to our premium
features such as viSNE. Contact [email protected] for more information about setting up an Enterprise
Cytobank.
(1) Click to open the Healthy Human PBMC 26 Surface Markers experiment, located in your inbox as a
public experiment.
(a) click on the “Clone FCS Files” and then
(b) click “OK” when asked if desired to make your own copy of the example data set.
The resulting page will verify that you successfully cloned the experiment and the new experiment has
(2) Click on “View Experiment Summary” to begin analyzing
your copy of this experiment.
(3) Click on “Edit Experiment Details” under the Actions panel
and change the experiment name to include your name and
‘viSNE tutorial.’
(4) Click the “Update Experiment” button to save these changes.
(2)
(1)
(3)
(4)
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Cytobank Guide to viSNE Analysis
Gating Preparation
(5) Click on “My Working Illustration” in the upper left corner and then click on “Gate”under the Populations panel.
Here is the gating page. This is the location where we’ll draw a few gates to clean up the data and remove debris.
(6) Change the x axis
to NA-191 and draw a
polygon gate around
the singlet population
– name the gate ‘Intact
cells’ and click “OK.”
(5)
(7)
(7) Double click in-
side the gate to make
Intact cells your active
population, change
the x axis to CD45, and
draw a new gate to
include only the CD45
positive cells and label
it CD45+.
(6)
(8) Once you are sat(8)
isfied with your gates,
click “Apply and Return” in the upper left
corner, and then click
“Back to Experiment.”
Running viSNE
(9) At the bottom of the Actions panel, click the
(9)
“Create viSNE Analysis” link and give your new analysis a name.
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Cytobank Guide to viSNE Analysis
Setting up the viSNE run
On the viSNE setup page, you’ll find three fields: Populations, Channels, and a down-sampling field.
(10) In this instance, click on
(10)
the “CD45+ population” under
Populations, click the selection
box next to the file and then
click “Done.”
Note: For this experiment, only
one file is present to be selected. In future experiments, you
can choose to run viSNE on
only a subset of available files.
(11) The down-sampling step will result in a cleaner viSNE plot and a faster run-time, but may not be
necessary if the files are small. For this tutorial, down-sample to 20,000 events by entering the number in
the “Desired Total Events” text box.
Note: If you’ve selected more than one population to run viSNE on, using the proportional downsampling technique will keep the relative levels of the populations the same so as not to alter their representation. Equal downsampling uses the same number of events from each population selected to build
the viSNE plot.
(11)
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Cytobank Guide to viSNE Analysis
(12) Click on “Choose” under the Channels field, select all
(12)
the CD markers, as well as HLA-DR and IgM and then click
“Done.”
(13) Click the “Run viSNE Analysis”v button. Once the analysis
is complete, the page will refresh to the gating interface and
you’ll receive an email with the link to your viSNE run.
Analyzing the results: gating on the viSNE
plot
(14) Once the analysis is finished, a second experiment is
created and linked to your original.
This new experiment has the two new viSNE channels that you can now use in gating and figures.
To see these channels, go back to the gating interface and change the axes to tSNE 1 and tSNE 2.
These channels work just like any other channel in Cytobank, however now a z axis is available which
you can use to color the events based on expression of any third marker, not just density.
(15)
(16)
(17)
(15) Each dot on the viSNE plot represents a single event from the original file.
(16) Each of the continents or populations present in a viSNE plot represent a common group of cells,
however there may be some markers that vary inside each population so feel free to split a population
with a gate, just like you would with traditional gating.
(17) Ensure that the x and y axes are set to tSNE1 and tSNE2, the plot type is Dot Plot (Stacked), and then
change the z axis to CD4.
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Cytobank Guide to viSNE Analysis
(18) Draw a polygon gate around the CD4 positive population and name it ‘CD4+’, and then continue
with the remaining populations (CD8 T cells, B cells, Monocytes, NK cells, etc).
(19) Click “Apply and Return” to return to the Work-
ing Illustration, and set the x and y axes to tSNE1
and tSNE2 respectively, and the z axis to Use Panel/
Channel values.
(20) Under the Populations figure dimension, click
“Choose” and select CD45+, then click “Done.”
(19)
(18)
(20)
(21) Under the Channels figure dimension, click
“Choose” and select CD3, CD4, CD8, CD16, CD19,
CD33, CD45RA, and CD123.
(22) Ensure the selected plot type is Dot Plot
(Binned) and then click the green link to update
the figure after clicking on the Illustration tab.
(21)
(22)
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Cytobank Guide to viSNE Analysis
(23) The resulting figure will show each continent of the viSNE plot with a defining channel highlighted.
(24) Save the figure by clicking in the Save Illustration As text box in the upper left corner and naming it
“viSNE phenotyping – tutorial”.
(23)
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