Transcriptome mapping using massive cDNA sequencing. (A) Whole-transcript sequencing. S. solfataricus ds-cDNA libraries are prepared from total RNA using random hexamers priming (stage 1). Resulting cDNA is sheared in random positions, and Illumina adapters are ligated (stages 2–3). Sequenced short reads are mapped to the genome (stage 4) to generate a transcript coverage map (stage 5). Shown is a 1.7-kb window of the genome, containing (brown arrows) four genes (right- and left-pointing arrows correspond to genes encoded on the forward and reverse strands, respectively). (Black bars) Sequenced reads, mapped to their respective positions on the genome. (B) 5′-End sequencing. An RNA oligonucleotide corresponding to the Illumina 5′ adapter is attached to exposed 5′ ends of total S. solfataricus RNAs (stage 1). cDNA is then synthesized using random DNA hexamers flanked by the Illumina 3′ adapters (stage 2). Transcripts are primed from the 5′ adapter, thereby generating strand-specific sequences matching 5′-ends of cellular RNAs (stage 3). Sequenced short reads are mapped to the genome (stage 4) to generate a transcription start sites (TSS) map (stage 5). Shown is the same region as in panel A. (Small arrows) Short reads mapped to their respective positions on the genome; (red) forward strand; (blue) reverse. Reads in the middle of genes might represent degraded/fragmented RNAs that present an exposed 5′-end.
