What main parameters should I consider when setting up a new RNA-seq experiment?

The main parameters to consider when setting up a new RNA-seq experiment include: replicates, sequencing coverage, multiplexing, enrichment/depletion, cDNA synthesis, and read length and type. One should consider how many replicates to include when doing the experiment. Technical replicates test the overall technology and workflow, while biological replicates are used to measure the significance of an observation. Sequencing coverage refers to the number of unique reads that include a given nucleotide in the sequence. The higher the read depth, the more likely the base can be identified correctly. Factors that affect sequencing coverage include genome size, gene expression levels and read length. The read length refers to the number of bases that will be sequenced from the end of a given transcript. Experiments for profiling known transcripts and small RNA molecules typically require shorter reads than experiments for assembling novel transcriptomes. Multiplexing is a procedure where multiple samples are mixed and run together on a single sequencing lane. The benefit of this parameter is that it allows a balanced block design to reduce risks such as PCR artifacts or cell flow effects. This in turn gives higher data quality. Enrichment/depletion involves using enzymes to rid of unwanted RNA species and enrich the desired RNA species for the experiment. During cDNA synthesis, the process of the reverse transcription of RNA to cDNA is carried out by using oligo (dT) primers or hexamers to prime the reverse transcriptase.