I am now in the last interview for a PhD application in which I was asked to come up with a strategy to study how a certain mutation in one of the RNA binding proteins affects the cryptic exon splicing in skeletal muscles. I did some reading and so far my plan is to take out the muscle(from an animal perfused with PBS and DEPC), separate the cells (using FACS or magnetic beads), extract RNA, perform bulk RNA seq, look for interesting candidates, and validate them with qPCR.

However, I never did FACS nor RNA seq before, so I am a bit fuzzy on the details and whether my strategy is good. For example, things like: do I need to make sure to separate only muscle cells of the muscles, does that add value or is it just extra work and money? Also, what kind of RNAseq is best to use to find mRNA containing unwanted cryptic exons (single nuclei vs bulk/ high depth?), what type of analysis tools are out there? How much time does such a project take? I would be grateful for any insights and ideas as discussing about it will help me understand everything better. Thank you!