Our in silico work has been fully funded via access to an HPC cluster with GPUs. The in vitro portion of our project is 3 stages: cell culturing, library preparation, and scRNA/scATAC sequencing.

The requested funding will cover the first stage (cell culturing) and the third stage (scRNA/scATAC-seq sequencing). The cost of the library preparation stage will be covered by other sources, including existing funds.

We will have enough for 6 samples (1 sample each for 3 timepoints, two experiments), with 6000 cells/sample sequenced, at a depth of 30,000 reads/cell. We remain open to adjusting these numbers should we find a better configuration or receive extra funding. All pricing information was determined in correspondence with McMaster University’s Genomic Facility. A more detailed budget breakdown of the three stages mentioned can be found in the lab note.

We have access to all other needed lab equipment and facilities at the University of Waterloo and Wilfrid Laurier University.

Additional Information

1. Detailed pricing information & cost breakdown can be found in the lab notes. A detailed bioinformatics plan can also be found in the lab notes which explains how our bioinformatics work will give us the answers to our questions.

2. One of the main authors of the “Derivation of a continuous myogenic cell culture from an embryo of common killifish, Fundulus heteroclitus” publication, Dr. Nguyen Vo, is a member of our project. Dr. Vo has developed an embryonic myoblast cell line (KFE-5) from killifish, which appears to be the first continuous fish myogenic cell line ever made. The cell line consists of two core phenotypes: mononucleated fibroblastic cells, and elongated myoblastic cells with the capability to differentiate into myocytic cells. Our scRNA/scATAC-seq data will help us reveal the pathways involved with the differentiation of the cells into these two phenotypes.

3. The researcher behind the single-cell analysis of bovine satellite cells project, Dr. Tobias Messmer, is an official advisor on our project.

4. Michael Saad, the only academic researcher who has done any level of transcriptomic analysis and successful cell line development for cultivated seafood, is an official advisor on our project.

5. Should our project be successful, we plan to extend our research to three more species: trout (Oncorhynchus mykiss), carp (Cyprinus carpio), and walleye (Sander vitreus). These species were selected based on many factors such as ease of access to organisms, commercial value, team expertise, and existing literature support. Our team will also benefit from having access to the University of Waterloo’s aquatic animal facility and a strong network of professors who specialize in fish biology.

6. Another future application that can be explored is using the data collected to guide the development of antibodies. These antibodies can then be used to isolate satellite cells or other desired cell types for the above three species.

7. Our sequencing will be outsourced because our university does not have single-cell sequencing capabilities and expertise. Our top choice is McMaster University’s Genomics Facility: https://genomics.healthsci.mcmaster.ca/services/10x-single-cell-sequencing/. Outsourcing will allow us to focus on our core expertise in bioinformatics/computation and fish biology. It will also help us save costs since we won’t need to build up the sequencing infrastructure from scratch. We have budgeted enough funds to run the entire experiment twice (from culturing to sequencing) to ensure the reliability of the results.