Soon I will be performing a cell type-specific differential RNA-seq experiment. We are able to sort ±2500 cells per sample. We are interested in differential expression between conditions, but not in SNPs, splice variants, modifications etc. We were wondering which method of RNA sequencing would be recommended? We have been suggested single end (3’ or 5’) and paired end, as well as different read lengths. Does anyone have experience with/ideas about what would work in C. elegans for this kind of experiment? Thank you.
I’ve used paired-end sequencing for bulk RNAseq (2000 cells). We’ve chosen this so we would also get information about possible oversequencing of the sample, using the UMI added to each mRNA molecule
Thank you for your reply. An additional question regarding your sample prep - in what conditions did you collect your cells, and how did you extract RNA from them for library prep? Cheers.
Sorry for the late reply: forgot to check the forum!
I’ve sorted my cells into Trizol (200 uL for 2000 cells), then continued to a chloroform extraction. Before the extraction, I add spike-ins and glycoblue