Total RNA of P. syringae DC3000, with (E+) or without (E−) Terminator exonuclease treatment was treated with tobacco acid pyrophosphatase (TAP) to convert transcripts to 5′mono-phosphate RNA and sodium periodate to block 3′ hydroxyl groups. Next, a 12-nucleotide RNA linker was ligated to available 5′ends, and RNA was reverse transcribed using a random primer that contained the Illumina adapter sequence. Single stranded cDNA was PCR amplified using the Illumina adapter sequences.

In the correlation plots, each point indicates the reads counts. (A). Scatter plot of TAP-only treated biological replicate 1 (E1−) compared to TAP-only treated biological replicate 2(E2−). (B) Scatter plot of Exonuclease treated biological replicate 1 (E1+) compared to exonuclease treated biological replicate 2 (E2+). Correlation scores (R²) for each comparison are shown.

Counts from the merged profiles (E12− and E12+) were paired in a coordinate and strand-sensitive manner at every location in the genome. The 3-way classification scheme was used to partition the pairs into subsets containing paired values from annotated (blue), opposite (green) and intergenic regions (red). All pairs containing one or two zero counts were discarded. Because the dynamic range of counts is so large, each value was incremented by 1 and log(10) of both values was calculated. Pairs from each of the three subsets were separately binned using a 100×100 grid. The grids were normalized by dividing the counts in every cell by the largest cell count in all three grids. Each grid was assigned a color (red, green or blue) with cell values establishing color intensity. The grids were then layered to produce a full-color density plot. The extremely high cell values in the vicinity of the origin were set to zero prior to normalization to boost the visibility of cells with small count values. This produces the black square in the lower left corner. The white line represents the threshold used to select the E12+Top.

Sequence logos of nine motifs generated by MEME [28]. MEME motifs are represented by position-specific probability matrices that specify the probability of each possible letter appearing at each possible position in an occurrence of the motif. The sequence LOGOS contain stacks of letters at each position in the motif. The height of the individual letters in a stack is the probability of the letter at that position multiplied by the total information content of the stack. The height of a letter indicates its relative frequency at the given position (x-axis) in the motif.

Composites of all of the annotated HrpL (A) and those PvdS (B) promoters exhibiting PvdS-dependent expression were constructed. Each promoter is represented below the X-axis by a red box and is oriented 5′ to 3′, left to right. Above the X-axis are spikes representing log(10) pseudocounts for captured 5′ ends at the corresponding genomic coordinates. The counts are derived from Supplemental Dataset S6. Green spikes are counts on the negative strand and blue spikes are on the positive strand. The promoters and their downstream regions are presented one after another with no intervening space. Promoters located on the negative strand are denoted with asterisks (*). The horizontal line just above Y = 2 is the cutoff (118 counts).

(A) represents an examples of putative antisense activity. (B) depicts an example of a putative new CDS and (C) is an example of a putative non-coding small RNA. The transcriptional profile (orange and purple) is shown above the annotated genome. An orange trace indicates transcription from left to right (on the positive strand) and a purple trace represents transcription from right to left (on the negative strand). In panel (A), the magenta colored misc_signal and highlighted sequence represents a putative promoter sequence. The nucleotides highlighted green are confirmed transcriptional start sites. IN panel (B) the blue CDSs represent EasyGene calls. In panel (C), the misc_signals (blue and green) correspond to predicted promoter sequences identified through the MEME analysis.