Or "Everything You Always Wanted to Know About RNA-Seq (But Were Afraid to Ask) Part 1"

Dear all, I am afraid I am too much of a data analyst and too little wet (never seen a flowcell in my life) to understand the real reasons why the library size varies (even by millions of reads!) between samples sequenced on the same flow cell. Isn't megareads per sample a parameter decided at the machine level? Does this variability only have to do with multiplexing different samples on the same lane or is there more to it (amount of RNA or cDNA, PCR efficiency, sequencer intrinsic features)? Can anyone enlighten me about the different factors that affect this parameter, i.e., sample-specific sequencing depth?

Thank you so much.

[ crossposted on Biostars: https://www.biostars.org/p/9563303/ ]