# Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in _E. coli_
## 3 Discussion
### sRNAs in Stationary Phase Biology  
Our work highlighted several sRNAs as potential regulators of stationary phase biology (Figure 2c). The number of differentially expressed sRNAs was higher than our initial prediction. In fact, several sRNAs candidates predicted to be deriving from coding regions were not detected in Nnorthern blot analyses (data not shown). Using a more stringent cutoff level for read counts may better recapitulate stationary phase enriched sRNAs. Within the top upregulated sRNAs, several candidates were validated by Nnorthern blot analyses (Figures 3a and 4a), suggesting that our biocomputational analysis captured the status quo of these transcripts in the stationary phase. By lowering the cutoff levels, we captured 1199 differentially expressed sRNAs in the stationary phase (Supplementary Table S4), of which the mRNA-derived _sRNA_vhfk_ was confirmed by  
Figure 6: Comparison of biocomputational methods for processing product identification. (**a**) Scheme showing processing product identification by APART [40]. The processing products were identified by sites of consequence, sharp, and reversed coverage shift (green circles). Other sites of sharp coverage shift were discarded (blue circles). Some processing products were accepted (green checkmarks), and others were discarded (red checkmarks). #Reads = number of reads. (**b**) Scheme showing processing product identification by Flaimapper [38]. The processing products were identified by peak detection on 5\({}^{\prime}\) and 3\({}^{\prime}\) end densities, followed by reconstruction of fragments. Red cross = discarded peak. #Ends = number of ends. (**c**) Scheme showing detection of overlapping processing products by STARPA. Overlapping processing products (red checkmark) could be discarded. To avoid this, STARPA fractionates reads by size first, then employs Flaimapper to identify processing products from each fraction. Northern blot analysis (Supplementary Figure S4). However, _sRNA_yhfk_ was lost from our analysis with the currently used more stringent cutoff. One potential solution to this problem is to add an extra step in STARPA, whereby reads are further segregated into new categories based on their biotype and then analyzed separately. For instance, reads deriving from rRNA regions are much more abundant than those derived from coding regions, and as such, a higher threshold for the former can be employed, and lower thresholds can be used for the latter. Predicted sRNAs differed between libraries with some overlap (Figure 2c), suggesting that these upregulated sRNAs belong to different classes of regulatory sRNA and may thus employ distinct mechanisms of action to establish or aid entry into the stationary phase. Our ribosome-associated sRNA libraries reported enrichment of _ssrA_ RNA that acted as tmRNA to release stalled ribosomes, a process more likely to occur upon nutritional starvation in the stationary phase [41]. As such, the upregulation of _ssrA_ in the stationary phase is crucial for bacterial survival under challenging environmental conditions. These same sRNA libraries predicted the enrichment of a _165 rRNA_ fragment in the stationary phase (FM_-NC_000913.2_3426703_81, Supplementary Table S3). We have previously demonstrated that this rRNA fragment is the result of _165 rRNA_ cleavage specific to the stationary phase and that the 30S small ribosome subunits carrying the fragmented _165 rRNA_ translate less efficiently than their exponential phase-derived counterparts [42]. Consequently, this reported processing product could represent a signal of ribosome shutdown in the stationary phase. Similarly, our analysis reported an abundant _235 rRNA_ fragment in the stationary phase (Figure 3a), whose function remains unknown, along with other rRNA-derived and ribosome-associated sRNAs predicted by STARPA (Figure 2c, Supplementary Table S3). These sRNAs might be signals of processing events in broader physiological responses and not regulatory sRNAs in themselves, such as the characterized _165 rRNA_ fragment.