Fig. 2Overview of RNAseq library synthesis. (a) For First Strand Synthesis, untailed hexamers (NSR Primers)
and SuperScript III Reverse Transcriptase synthesize cDNA from the RNA. The products of this reaction are
RNA:DNA heteroduplex molecules. (b) For Second Strand Synthesis, the first strand cDNA product is converted
to dsDNA by RNase H-mediated nick translation. RNase H introduces breaks in the RNA template strand, and
then Pol I synthesizes DNA from these sites by using the RNA as a primer. The 5^0 –3^0 exonuclease activity of Pol
I removes RNA during nascent strand extension. DNA ligase then patches any gaps initiated from the different
priming sites. The products of this reaction are dsDNA fragments that contain short 3^0 overhangs. (c) End
186 Charlotte D. Majerczyk