dataesr/scientific-paragraphs-categorization huggingface dataset:
Acknowledgements The authors would like to thank  Paula Freire-Pritchett  for help with data analysis,  Simon Andrews ,  Steven Wingett ,  Felix Krueger  at the  Babraham Institute's Bioinformatics facility  for help with data management and initial processing,  Kristina Tabbada  and  Clare Murnane  ( Babraham Institute sequencing facility ) for Illumina sequencing,  Arthur Davis  ( Babraham Flow Facility ), the  Wellcome Trust-Medical Research Council ,  Institute of Metabolic Science, Metabolic Research Laboratories, Imaging core ,  Wellcome Trust   Strategic Award  [ 100574/Z/12/Z ] for technical assistance and the  Cambridge National Institute for Health Research Biomedical Research Centre Cell Phenotyping Hub  for cell sorting,  Alison Finigan  ( Cardiovascular Medicine Division, University of Cambridge ) and  Mark Lynch  ( Fluidigm ) for technical assistance,  Hashem Koohy  for advice on random forest analysis and all members of the  Spivakov and Jørgensen labs  for helpful discussions.
RNA extraction was then performed with the All-in-One Purification Kit (Norgen), allowing the separation of the RNA within a small RNA (<200 nt) and a large RNA (>200 nt) fraction. The "large RNA fraction" (>200 nt) has been used for RNA-Seq, even though our data clearly indicates that all small RNAs (except tRNAs) can be resolved by RNA-Seq within the "large RNA fraction" (Figures  3, 11 ). OMV-associated RNA was further concentrated using a Zymo Research RNA clean up and concentrator kit (including On-column DNaseI treatment) up to a volume of 6 µl (corresponding to 1. 5 l of original culture). The extraction of intracellular RNA was also performed with the All-in-One Purification Kit (Norgen), followed by a DNaseI treatment. 1 µl of RNA was qualitychecked with an Agilent 2100 Bioanalyzer.
Libraries were diluted to 4 nM each, pooled, denatured and sequenced 2 × 80 (Low OD) or 2 × 75 (High OD) cycles on a NextSeq500 (Illumina) according to the manufacturer's instructions. For each experimental condition, three biological replicates were sequenced. The raw sequence libraries are deposited in the BioProject database under the experiment accession number PRJNA438234, available at https://www. ncbi. nlm. nih. gov/bioproject/438234.
RNA Sequencing Annotation Raw RNA 75 nt or 80 nt paired-end sequencing reads from each condition were first trimmed and quality filtered using the trim-fastq. pl script from PoPoolation package with the following parameters -quality-threshold 20 -fastq-type sanger -minlength 35  (Kofler et al. , 2011). The Salmonella genome and plasmid sequences and annotations (in GFF3 format) were downloaded from NCBI (genome accession number AE006468. 2 and plasmid accession number AE006471. 2). Samtools were used to merge the bam files  (Li et al. , 2009). Tophat2 was used to map the trimmed paired-end and single-end reads to the Salmonella genome  (Kim et al. , 2013). Moreover, small RNA sequences from  (Srikumar et al. , 2015)  were mapped to the LT2 genome. Only sequences that had 100% sequence identity over the full length on LT2 strain were retained.
Count Normalization, PCA and ANOVA Normalization among samples and conditions was performed using the DESeq2 package  (Love et al. , 2014) , i. e. , results were normalized for library sizes but not for gene length as the purpose of our analysis was to compare the expression of identical genes in different conditions. Moreover, rRNA was removed in silico from all datasets (otherwise stated) to avoid potential biases in the comparisons between intracellular and OMVassociated RNAs as they were experimentally removed from intracellular fractions only. Principal component analyses (PCA) were obtained with the plotPCA function from DESeq2. Analysis of variance (ANOVA) was performed with subsequent post hoc analysis to test if significant differences in relative amounts of exported RNA classes between the different culture conditions exist, using Tukey's range test from the multcomp package  (Hothorn et al.
PCR Validation To confirm results obtained by RNA sequencing and to show that RNAs of interest are present in or on OMVs in full-length, PCR validation was performed for distinct RNAs (Supplementary Dataset C) on independent replicates from high OD conditions. cDNA Library Synthesis New S. Typhimurium intracellular samples and purified OMV fractions were obtained from SPI-1ind, SPI-2ind, and Control conditions at high OD, with two biological replicates per condition. Intracellular and OMV-associated RNA was obtained as previously described with the following differences: the initial culture volume was 0. 75 l per replicate instead of 1. 5 l; RNA extractions were performed using TRIzol Reagent (Invitrogen); cDNA libraries were prepared from 1 µl of OMV-associated RNA or 100 ng of intracellular RNA with the SuperScript III First-Strand Synthesis System (Invitrogen) using random hexamer primers according to the manufacturer instructions and without further cDNA fragmentation step.
Full Length RT-PCR and Visualization of the Amplicons Polymerase Chain Reaction was performed using Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific). A set of primers was designed to amplify the totality of the targeted Open Reading Frame (from start to stop codon), or the totality of the selected ncRNAs (list of targets and primers available in Supplementary Dataset C). 0. 5 µl of cDNA library was used for each 20 µl PCR reaction as template, together with 1 µM of primers. Intracellular libraries were used as positive control, and cDNA synthesis reactions were performed on OMV-associated RNA without adding reverse transcriptase (RT) as a negative control to check for the presence of residual genomic DNA. Three different Touch-Down PCR programs were used, depending on the target length and differed only by their extension time (15 s per cycle for small targets under 500 bp, 45 s for medium targets between 500 and 1500 bp, and 70 s for large targets above 1500 bp).
RNA Is Associated With S. Typhimurium OMVs RNA sequencing (RNA-Seq) was performed on the RNA isolated from OMVs secreted by S. Typhimurium in order to study their RNA composition. A significant number of transcripts was found in OMVs under all conditions (control Low and High OD, SPI-2ind Low and High OD, SPI-1ind High OD). Reads were mapped to the S. Typhimurium LT2 genome and pSLT plasmid and annotated using NCBI, and sRNA supplemental annotations from  (Srikumar et al. , 2015). Basic statistics of the RNA-Seq data are displayed in Supplementary Dataset A for low and high OD samples. Briefly, intracellular samples had roughly all around 10 million reads mapped to the target genome. For OMV samples, the number of reads and mapping percentages were lower, especially for SPI-1ind samples. The main contaminating RNAs mapped to Agrobacterium, Rhizobium, and Achromobacter taxa, which are common contaminants originating from column purification kits, when dealing with low abundance samples  (Susilawati et al.
The Proportion of RNA Classes Associated With the OMVs Varies According to the Environmental Condition When analyzing the RNA classes present in OMV-associated fractions, it became clear that every class of transcript is exported, including rRNAs, mRNAs, tRNAs and others ncRNAs (Figure  3  and Supplementary Dataset B). One can observe that for all but the OMVs originating from SPI-1ind, the vast majority of exported RNAs were rRNAs, as it has already been observed in other species [e. g. , in Escherichia coli, see  (Blenkiron et al. , 2016) ]. However, in the case of SPI-1ind grown bacteria, the proportion of rRNA associated to OMVs was much lower, down to only 12. 1 ± 15. 2%. Concomitantly, we observed an increase in the mRNA relative amount up to 85. 9 ± 15. 1%. Despite the variance observed between triplicates, both of these values were significantly different from their respective counterpart in other conditions as determined by Tukey's range test (Figure  3 ).
Thus, it seems that the balance between rRNAs and mRNAs depends on the environmental conditions (pseudogenes followed the same trend than mRNAs). This result needs to be put in perspective, taking into account the lower sequencing depth obtained for the SPI-1ind OMV samples (Supplementary Dataset A). On the contrary, no significant differences were detected concerning tmRNA, tRNAs, and others ncRNAs, which appear to have been exported the same way in all conditions (see all ANOVA and Tukey's test results in Supplementary Dataset B). Concerning tRNAs, we found a limited relative amount of tRNAs (<0. 2% for all conditions). These were probably exported, but mainly found in the small RNA fraction (<50 bp) and therefore would have probably been more visible in a small RNA-Seq library, which was not included within the present study  (Ghosal et al.
OMV-Associated RNAs Are Either Full Length, Processed and/or Degraded As some exported RNAs appeared to be complete transcripts according to their coverage profiles, we designed a set of primers to validate their integrity experimentally by PCR amplification. The full coding-sequence of 20 mRNAs and the totality of 10 ncRNAs sequences were targeted and amplifications were performed on high OD condition samples (Supplementary Dataset C). 12 RNAs were found to be amplified in their fulllength from our cDNA libraries in at least one condition: 7 ncRNAs (IsrB-1/2, ffs, IsrA, SsrS, CsrC, 10Sa, rnpB) and 5 mRNAs [pSLT035, STM0277 also called tae4, sseB, STM0972 (sopD2), STM2606] (Figure  5 ). Full-length mRNAs were amplified in only one out of the two replicates analyzed (for instance, sseB and STM0972 in the case of SPI-2ind High OD).
STM0277 was also detected in SPI-2ind High OD but not in SPI-1ind, whereas a greater number of reads was detected in OMVs from the later by sequencing. Interestingly, we observed a tendency toward the amplification of the smaller sized RNAs compared to the larger ones (Figure  5  and Supplementary Dataset C). This trend might be due to a higher degradation predisposition of larger RNAs compared to small RNAs, which are usually more structured and therefore more stable. Especially, protein-associated ncRNAs such as ffs (SRP RNA binding to Ffh and routing nascent proteins to the membrane), rnpB (RNA catalytic component of the RNaseP ribozyme involved in pre-tRNAs maturation), SsrS (6S RNA that binds and regulates the σ 70 transcription factor) or 10Sa (tmRNA associated to SmpB) were remarkably conserved in OMVs, in complete agreement with the RNA sequencing results (Figures  4, 5  and Supplementary Figure  S8A ).
However, this association might also occur when these are bound to their partner proteins explaining their stability. Note that in the same digestion conditions, intracellular FIGURE 4 | Visualization of intracellular and OMV-related read coverage plots of distinct sRNAs in distinct culture conditions. RNA coverage from sequencing data was visualized with the Integrative Genomics Viewer software (v2. 4. 8) using default parameters  (Robinson et al. , 2011). Each plot represents the raw number of reads mapped along the observed sequence, automatically scaled relatively to the highest number of read existing in this portion of the genome. The three plots in dark colors at the top of each window represents the sequencing coverage of OMV-associated fractions, in biological triplicate for each condition.
On the contrary, the three plots in lighter colors at the bottom of each window show the coverage for the same RNA but from the corresponding intracellular fractions. The arrows under each window precise the genes positions and orientations, according to the data extracted from the Salmonella LT2 genome annotation (NCBI accession number AE006468. 2) or pSLT plasmid annotation (NCBI accession number AE006471. 2). The identical coverage patterns observed in both fractions for the displayed genes stand for a native export through OMVs without specific processing or degradation. RNA or protein extract was almost completely digested and only rnpB gave a low signal by RT-PCR after RNase treatment (Supplementary Figures  S5B, C ). Some -RT controls showed a remaining DNA contamination (Supplementary Figure  S9 ) despite the DNAseI digestion step before cDNA synthesis.
Therefore, we performed qPCR on +RT and -RT samples for several genes and confirmed that the amount of cDNA was greater in +RT than in -RT for ffs, CsrC, rnpB, sseB, and STM0972 (sopD2) (Table  1 ). For sseB and STM0972, both replicates were positive contrary to the full-length PCR, indicating that the transcripts were present but not in their native form or the end-point PCR was not sensitive enough to detect them on a gel. In contrast to the genes highlighted above, no full-length transcripts associated to OMVs could be detected for the following genes: STnc440, STM0278, STM1026, pagC, spvD, STM0292, ydgH, sseI, AmgR, sipC, nanH, gogB, sseC, sipB, sipA, STM0289, slrP, and STM0291 (Supplementary Dataset C).
Two biological replicates for SPI-1ind, SPI-2ind High OD and Control High OD conditions are visualized. Primers can be seen in Supplementary Dataset C, -RT controls are displayed in Supplementary Figure  S9  and a positive control of full-length amplifications performed on intracellular RNAs is visible in Supplementary Figure  S10. FIGURE  6  | RNase protection assay of selected protein-associated sRNAs isolated from OMVs. OMV isolation and enzymatic digestions were performed from SPI-1ind condition as described in Section "Materials and Methods (see section "OMV Purification and RNA Extraction"). " Crude OMV preparation was divided in four fractions. One was kept untouched (Untreated). The second was subjected to the same protocol than the third and fourth fraction without any enzyme added (Control).
For two given technical duplicates, if the difference between the average of +RT C t and the average of -RT C t is equal or superior to 3, the difference is considered as significant (eight times more cDNA than residual gDNA in the sample). n. s. , no signal after 45 cycles, meaning no amplification was detected. Water controls always led to n. s. Primers information is displayed in Supplementary Dataset C. in/on OMVs as described and explained in section "Native and Processed and/or Degraded RNA Co-exist Among OMV-Associated RNAs. " As, however, this remained true for all the different culture conditions, we performed pairwise comparisons between intracellular and OMV-associated RNAs among our five culture conditions, in order to obtain a global overview of RNA sequences which were differentially expressed among the different culture media.
As 10Sa binds other proteins for its activity, and among them elongation factors or ribosomal proteins, its OMV association could be favored by the presence of the elongation factor EF-G in the vesicles  (Bai et al. , 2014; Himeno et al. , 2014). However, no trace of its main protein partner SmpB has been reported to date. In order to define more precisely the OMV-enriched transcripts in comparison to their intracellular counterpart, a differential expression analysis with the DESeq2 package  (Love et al. , 2014)  was performed. We chose to select RNAs with a log 2 Fold Change (logFC) (OMV/intracellular) ≥ 2, a p-value < 0. 05 (Welch's t-test), and an averaged number of reads per condition ≥ 5 in order to ensure a stringent selection of "enriched RNAs. " Henceforth, all RNAs that fulfill the aforementioned criteria are referred to as enriched RNAs, whereas we refer to exported RNAs when a given RNA is associated with OMVs, but not necessarily enriched compared to its intracellular counterpart.
In each media condition, a significant number of transcripts was indeed selectively exported (Figure  8 ). In total, we found 1151 enriched transcripts in Control Low OD condition, 763 in SPI-2ind Low OD conditions, 881 in Control High OD condition, 643 in SPI-2ind High OD condition and 1196 in SPI-1ind condition (detailed lists are available in Supplementary Dataset D). A smaller core set of 193 RNAs was enriched in all conditions tested, with the notable absence of ncRNAs in this set, these being mainly found in condition-specific groups. However, these group sizes were very low compared to the totality of the RNA cargo, which originated from 4670 genes in total, from which 2217 were found in all the conditions tested (Supplementary Figure  S6A ). This difference may underline the dual nature of OMV-RNA association, reflecting the intracellular state of the transcriptome through global packaging, but also containing specific sets of read fragments mapping to distinct RNAs that depend or not on the current environmental state.
Virulence Effectors Related RNAs Are Among the Selected Cargo for Export As we validated the association of several full-length virulence-related mRNAs with OMVs (Figure  5 , Table  1  and Supplementary Dataset C) and as most of the effectors were covered over the whole length of their gene (Supplementary Figures  S11A-E ), we looked at the more than 40 effectors known to be secreted by Salmonella during invasion and infection  (Niemann et al. , 2011; Yoon et al. , 2011a; Kidwai et al. , 2013; LaRock et al. , 2015). Intracellular expression, OMV-exported and enriched effectors are displayed in Figure  9. As expected, the intracellular expression of secreted molecules was linked to their known export route: SPI-1 translocated effectors were more expressed in non-SPI-2ind conditions and vice-versa (Figure  9A ).
However, even if almost all effectors' mRNAs were found associated with OMVs in every condition, the OMV export route appeared to be more important when the corresponding T3SS (Type 3 Secretion System) was absent (Figure  9B ). This was confirmed by the analysis of enriched effectors RNAs, where a distinct pattern did exist (Figure  9C  and Supplementary Dataset E): SPI-1 translocated effectors seemed enriched in/on vesicles produced in SPI-2ind conditions (i. e. , sip genes), whereas SPI-2 translocated ones were enriched in/on OMVs originating from non-SPI-2ind conditions, i. e. , Control and SPI-1ind (i. e. , some sse transcripts). Interestingly, we also identified prophage-encoded effectors. For instance, GogB is a secreted effector originating from Gifsy-1 prophage that can be translocated through both the SPI-1 and SPI-2 T3SS, but which is mainly expressed under SPI-2 control by ssrB  (Coombes et al.
, 2011b). Interestingly, a number of SPI-6 mRNAs were found to be exported and enriched in/on OMVs (Figure  10 ). In this case, an increased intracellular expression caused a greater export by vesicles, as it was clearly visible in Control conditions at high OD and SPI-1ind. The in vitro expression level of SPI-6 is generally low, as reported earlier  (Mulder et al. , 2012). However, OMV-association apparently occurred in all conditions, and to a higher extent in non-SPI-2ind conditions (Figure  10B ). Interestingly, the same was observed concerning the enrichment in OMVs, showing a correlated expression and enrichment in vesicles for most of the SPI-6 transcribed RNAs (Figure  10C  and Supplementary Dataset F). The coverage FIGURE 8 | Repartition of enriched RNAs in/on OMVs isolated from the different analyzed in vitro culture conditions.
UpSet plot representing the number of enriched RNAs shared between different conditions. One unit represents one expressed gene. The graph was generated using the UpSet R package. Enriched RNAs were determined using DESeq2, calculating differential presence between intracellular and OMV-associated fractions for each condition. Expressed genes with an average number of reads over 5 for a given set of triplicates, a log 2 Fold Change over 2, and a corresponding adjusted p-value under 0. 05 were selected. ncRNA class attribution is defined from the Salmonella LT2 genome annotation (NCBI accession number AE006468. 2) or pSLT plasmid annotation (NCBI accession number AE006471. 2). Moreover, small RNA annotations from  Srikumar et al. (2015)  were mapped to the LT2 genome. Only sequences that had 100% sequence identity over the full length on LT2 strain were kept.
Identical datasets were used for the generation of Figure  8  and Supplementary Figure  S6B. data of the mapped reads was relatively constant between intracellular and OMV-associated fractions (Supplementary Figures  S12A, B ). SPI-6 in S. Typhimurium encodes for a Type 6 Secretion System (T6SS) that is involved in eukaryotic cell infection  (Mulder et al. , 2012). However, the role of the T6SS is not limited to virulence toward eukaryotes. Accumulating evidence demonstrates that the T6SS is mainly involved in interbacterial intoxication, targeting competitors by contactdependent transport of bacteriocins from a donor cell to a recipient cell  (Durand et al. , 2014; Russell et al. , 2014). Interestingly, one can note that the peptidoglycan hydrolase mRNA tae4 (STM0277: validated as being present as a full-length transcript within OMVs: Figure  5 ) and its cognate immunityprotein mRNA tai4 (STM0278) were only moderately present in the intracellular fraction (Figure  10A ) under the conditions studied, but their mRNAs were enriched in/on OMVs purified in SPI-1ind and Control conditions at high OD (Figure  10C ).
Some sRNAs Involved in Virulence Are Also Enriched in/on OMVs As we could experimentally validate the presence of several fulllength sRNAs (Figure  5  and Supplementary Dataset C) and as we have seen in Figure  8  that a smaller proportion of these latter was also enriched in/on OMVs in a condition-dependent manner, we analyzed in more detail these sRNAs (Figure  11  and Supplementary Dataset G). The majority of them was found in the SPI-2ind and SPI-1ind cases, thus linked to virulence-like conditions. An enrichment in/on OMVs was mainly observed in high OD conditions; with only a few transcripts enriched at low OD. If these were exported in their native form through OMVs, they might be involved in pathway regulation in surrounding cells up-taking OMVs. Traces of almost all pathogenicity-island encoded sRNA were found in OMVs (Supplementary Figure  S13 ).
This sRNA is controlling amino-acids uptake and has also a demonstrated positive effect on bacteria resistance to low pH in E. coli, which is a physiological condition encountered by Salmonella in macrophages  (Jin et al. , 2009; Sharma et al. , 2011). Others sRNAs involved in virulence were also found in/on OMVs, but without specific enrichment regarding their intracellular presence. For example, CrsB and CsrC (the later validated experimentally to be internalized as a full-length transcript in OMVs by PCR: Figures  5, 6 , Table  1 , and Supplementary Dataset C) were among the most exported sRNAs through OMVs, and the most abundant intracellular sRNAs (Supplementary Figure  S13 ). These sRNAs control the translational regulator CsrA that regulates SPI-1 genes and the expression of flagella components, playing a central role in virulence  (Fortune et al.
, 2006; Timmermans and Van Melderen, 2010). The 6S RNA, SsrS (validated experimentally to be internalized as a full-length transcript to OMVs by PCR: Figures  5, 6, Table 1 , and Supplementary Dataset C), was also highly expressed inside the cells and associated with OMVs. It is necessary for acidic-environment adaptation and epithelial cell invasion  (Ren et al. , 2017). STnc1480 is a PhoP/Q and SlyA regulated sRNA whose role during the intracellular life of Salmonella has been suggested, and was found in OMVs in a high amount  (Colgan et al. , 2016). SgrS, a repressor of sugar uptake that also represses the SopD secreted virulence factor  (Papenfort et al. , 2012; Balasubramanian and Vanderpool, 2013) , was also exported in all conditions.
However, the confirmation of the export of full-length transcripts opens the door to numerous possible functional implications in cell-to-cell communication. to the whole Eco-Systems Biology group, as well as to Esther Nolte-'t Hoen for fruitful discussions. We also acknowledge the support and the use of resources of the French Infrastructure for Integrated Structural Biology FRISBI ANR-10-INBS-05 and of Instruct-ERIC for TEM imaging.