Setting Up an RNA-seq Experiment

HubSpot Video


This tutorial video gives you a step-by-step guide to setting up your bulk RNA-seq experiment. From giving your new project a name to setting up your comparison groups. If you prefer, you may also review our step-by-step guide below. 


Step-by-step guide: 

1. Create an experiment in ROSALIND by selecting "Add New Experiment" to an existing project folder or by selecting "Add New Project". 


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2. Enter a Title and Description for your Project.

3. Select "Design a New Experiment" from the menu below. 


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4. Enter a Title and Description for your Experiment. 

5. Select your Method and Experiment Type from the drop down menus. 

6. Select the species of your samples and the genome build from the dropdown menu. 

7. Specify the kit that was used for your samples from the dropdown menu. Select "Add Kit" to confirm. 

8. Specify if any of your samples are replicates of one another. 

9. Specify attributes for each of your samples or upload an attribute file. You can find further instructions on how to create an attribute file here


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10. ROSALIND provides you with a sample sheet based on your specified attributes. You may make any changes you need directly on the sample sheet or, if all your attributes look correct, you can select "Next" to continue. 


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11. You may next set up your comparisons by selecting "Create New Comparison" and then dragging and dropping different attribute values into the condition and control boxes on the right hand side of your screen. This is an optional step as you may create an unlimited number of comparisons before or after your experiment has been launched.  


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12. Upload your FASTQs by dragging and dropping your FASTQ files onto ROSALIND. Once uploaded, your experiment will begin to launch! See below for more detailed instructions for how to upload when you have multiple files per sample.

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Uploading Multiple FASTQ Data Files For One Sample

There are three common reasons why you may have more than one FASTQ data file per sample. These scenarios are described below, along with simple instructions on how to upload successfully.

1) You may have paired-end data

These files typically have R1 and R2 somewhere in the file name, which stand for Read 1 and Read 2, or are sometimes referred to as Left Read and Right Read. Two files correspond to one sample.





ROSALIND can easily handle paired-end data. Select paired-end from the toggle on the upload screen, and upload your R1 file to the Left Read (R1) box, and your R2 file to the Right Read (R2) box.


2) You may have multi-lane data

These files typically have something like L001 or L002 in the file name. Multilane data could have anywhere from 2 to 8 fastq data files per sample.





You can add multilane data to ROSALIND by uploading multiple files at once. Select all the files that correspond to each sample, and drag and drop them together into the appropriate upload box. Or, click on an upload box and select all the files that correspond to each sample.


3) You may have paired-end data that is ALSO multi-lane

These files typically have something like L001 or L002 in the file name. Multilane data could have anywhere from 2 to 8 fastq data files per sample.







Use a combination of the two upload instructions above. Upload all files for R1 to the R1 upload box, then upload all files for R2 to the R2 upload box.