Overview of a typical RNA-Seq experiment. RNA is initially fragmented (1) followed by first-strand synthesis priming (2), which selects the 3' fragment end (in transcript orientation), to make single stranded cDNA. Double stranded cDNA created during second-strand synthesis (3), which selects the 5' fragment end, is then size selected (4) resulting in fragments suitable for sequencing (5). Sequenced reads are mapped to opposite strands of the genome (6), and in the case of known transcript or fragment strandedness, the read alignments reveal the 5' and 3' ends of the sequenced fragment (see Supplementary methods in Additional file 3). All arrows are directed 5' to 3' in transcript orientation.