# Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in _E. coli_
## 2 Results
### Validation of Selected Candidates  
Deep sequencing of the small transcriptome of _E. coli_ identified several small ncRNA candidates that were differentially expressed during the stationary phase compared to exponentially growing cells. Highly expressed candidates from the stationary phase \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Description** & **LB Total** & **MOPS Total** & **LB Ribosome** & **MOPS Ribosome** \\ \hline Downregulated & 1024 & 1098 & 323 & 299 \\ Not significant & 1671 & 1574 & 1797 & 1909 \\ U upregulated & 393 & 416 & 273 & 185 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Differential expression analysis. Table showing the number of downregulated, unchanged (not significant), and upregulated sRNAs in total RNA libraries and ribosome-associated RNA libraries for both lysogeny broth (LB) and morpholinepropanesulfonic acid (MOPS) growth media. All numbers were based on differential expression analysis in the stationary phase in comparison to the exponential phase. Figure 2: STARPA revealed over 12,000 sRNAs. (**a**) Venn diagram showing the number of identified processing products in total and ribosome-associated RNA libraries, with a cutoff threshold of 10 reads per million or higher. (**b**) Pie charts showing the percentage of identified RNAs by biotype for total and ribosome-associated RNA libraries. (**c**) Venn diagram showing the number of upregulated processing products in the stationary phase, in total, and ribosome-associated RNA libraries deriving from cells grown in LB and MOPS. samples (Supplementary Table S3) whose sequences mapped with defined read stacks on the reference genome were selected for subsequent experimental validation. Northern blot analyses confirmed the abundant expression of some candidates in the stationary phase (Figure 3a), belonging to distinct classes of identified transcripts. Interestingly, some of the top enriched stationary phase-specific sRNAs were tRNA-derived RNA fragments (tRFs). Northern blot analyses confirmed the enrichment of these transcripts in the stationary phase (Figures 3a and 4a). Figure 3: Validation of identified sRNAs. (**a**) Northern blot analysis of three candidates: _sRNA_35_ (FM_+NC_000913.2_4541696_34), _235 rRNA_ fragment (FM_+NC_000913.2_3942854_40), and 5′-_tRF_*_17_ (FM_+NC_000913.2_3944979_33). _55 rRNA_ was used as a loading control. Zero point four and 2 were optical density measurements, h = hours, d = days. tRF = tRNA derived RNA fragment. Predicted sizes are displayed on the right in nt (nucleotides). (**b**) Northern blot analysis of candidates in A. P100 = pellet enriched for ribosome-associated RNA. S100 = supernatant containing non-ribosome associated RNA. (**c**) RT-qPCR mean and SEM for _sRNA_dps_ (FM_-NC_000913.2_0848114_53), _sRNA_bola_ (FM_+NC_000913.2_0453657_58), _sRNA_osmE_ (FM_-NC_000913.2_1820262_45), and _sRNA_ythfg_ (FM_-NC_000913.2_3489623_47) in the stationary phase (two biological replicates each). \(\log_{2}\) fold change was based on comparison with exponential phase samples for both conditions: Total S = Total RNA samples in the stationary phase. P100 S = ribosome-associated RNA in the stationary phase. To validate enrichment of some transcripts in ribosome-associated libraries, northern blot analyses were carried out on RNA samples extracted from ribosome enriched pellets (P100) after ultracentrifugation and corresponding supernatant fractions (S100). As expected from the differential sRNA expression analyses, _sRNA_35_ and _tRF\({}^{t\text{t}PT}\)_ were enriched in non-ribosomal fractions, whereas a _235 rRNA_ fragment was abundantly retained in ribosome fractions in a growth phase-dependent manner. Overall, tRFs appeared to be predominately non-ribosome bound in _E. coli_ (Figures 3b and 4b). Our sequencing results showed that a large subset of differentially expressed sRNA derive from mRNA coding regions (Figure 2b, Supplementary Figure S3).