Learn to set up a Single Cell experiment from a Seurat RDS Object with this step-by-step walkthrough.
Step 1: Create a new Project folder by clicking . Enter a Title and Description (both can be edited at a later time).
Click the button to proceed and automatically create a new Experiment.
Alternatively, click on an existing Project folder to open it. Then create a new Experiment by clicking . Click the button to proceed.
Step 2: Select "Design a New Experiment" to access all available assay workflows, then click Next.
Step 3: Enter an "Experiment Title" and "Description" (both can be edited at a later time), then click Next.
Step 4: If creating an Experiment from a Seurat RDS object, use the "Method (Technology)" drop down menu to select "Single Cell Transcriptomics". From the "Experiment Type" drop down menu, select "scRNA-seq (rds File)". Then click Next.
a)Click the Single cell object file upload box ("BROWSE OR DROP FILE") to navigate to and select the input file, or drag and drop the file into the box to begin upload.
b) Select your Sample Identifier: this is the column header for your samples column from your RDS file
c) Optionally, a completed Attribute File can be uploaded at this stage. If skipped, upload and creation of an Attribute File will occur later in the workflow.
d) Select your "Organ or tissue type" for ROSALIND Cell Type Prediction.
Note: Selecting "Other" will not opt out of running ROSLAIND Cell Type Prediction
Step 6: Select the Species and Genome Build from the dropdown menus. ROSALIND offers genome builds from NCBI and Ensembl. Unless comparing the experiment to historic data, it is recommended to select the most recent build from your preferred source (NCBI or Ensembl). Click Next.
Step 6: Specify sample attributes by typing the attribute categories into the "Enter Attributes" line.
After entering attribute categories, download an attribute file (.CSV format) by clicking . Within the file, attribute values can be manually assigned to each sample. The file can then be uploaded into ROSALIND by clicking . Further instructions for attribute file creation can be found here.
Step 7: ROSALIND displays a sample sheet to review for accuracy. Once reviewed,
Step 8: Comparisons can be configured now, prior to launching the analysis, or after the experiment has been processed and QC has been reviewed. Comparisons created now will be at the sample level. Single cell level comparisons can be created after experiment launch and processing. Comparison creation is identical between levels.
To create a new comparison now, click . Comparisons can be created at the replicate group level (using the attributes) or individual sample level (using the sample names) by dragging and dropping different attributes or samples into the condition and control boxes on the right side of the page. Combinations of two or more different attributes can be used to create comparisons with specific subsets of samples. In the example image below, a comparison is set up across two treatment groups (Radiation vs Control) at a specific time point (1 hour). Alternatively, the comparison could be set up specifically at only the 48 hour time point, or including both time points simultaneously.
Additionally, covariate correction can be added to the statistical model using the "SELECT COVARIATE" button. Click here to learn more about covariate correction.
Click the button to save the created comparison. Once all comparisons have been saved, click Next.
Step 9: The experiment will automatically begin processing for launch after comparison creation is complete. Do not navigate away from the launch page until your experiment has successfully launched.