For long-read de novo genome assembly, if I want to include short-read RNA sequencing data to perform annotation, how much sequencing do I need to include?

It depends on your desired outcome. Minimally, we’d recommend 50M-100M reads. For more confident annotations, ~200M-400M reads. For performing high-quality annotations with multiple tissue, we’d recommend a total of 500M-1B reads, with each tissue type receiving ~50M-100M reads.