Impact of ribosomal depletion, PCR ramp rate, and fragment length on RNA-seq

Coverage of short-read RNA-seq is highly non-uniform across transcripts even in genes with only one expressed isoform, contrary to biological expectation. We investigate the impact of several library preparation factors on the non-uniformity of coverage. Specifically, a mouse liver sample is prepared under varying ribosomal depletion (PolyA / rRNA digestion / rRNA pull-down / NoSelection), PCR ramp rate, and fragment length along with one heart sample without selection. One mouse liver sample was extracted and then prepared under several protocols. Each combination of selection method (PolyA / rRNA pull-down / rRNA digestion), PCR ramp rate (slow/medium/fast), and fragment length (short/medium/long) was prepared. In addition, libraries without any selection or depletion of rRNA were prepared ("NoSelect") in triplicate in two batches to increase the amount of mRNA reads. Finally, one heart NoSelect sample was prepared.