This work was supported by the &#8220;Agence Nationale de la Recherche&#8221; (ANR, Grant ANR-16-CE11-0007-01, RIBOSTAPH, and ANR-18-CE12- 0025-04, CoNoCo, to PR). It has also been published under the framework of the labEx NetRNA ANR-10-LABX-0036 and of ANR-17-EURE- 0023 (to PR), as funding from the state managed by ANR as part of the investments for the future program. DL was supported by the European Union&#39;s Horizon 2020 research and innovation program under the Marie Sk&#322;odowska-Curie Grant Agreement No. 753137-SaRNAReg. Work in E. Mass&#233; Lab has been supported by operating grants from the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), and the National institutes of Health NIH Team Grant R01 GM092830-06A1.

1. Buffers and media
<ol>
	<li>For MAPS experiments, prepare the following buffers and media: 
	- Buffer A (150 mM KCl, 20 mM Tris-HCl pH 8, 1 mM MgCl2 and 1 mM DTT) 
	- Buffer E (250 mM KCl, 20 mM Tris-HCl pH 8, 12 mM maltose, 0.1% Triton, 1 mM MgCl2 and 1 mM DTT) 
	- RNA loading buffer (0.025% xylene cyanol and 0.025% bromophenol blue in 8 M urea) 
	- Brain Heart Infusion (BHI) medium (12.5 g of calf brain, 10 g of peptone, 5 g of beef heart, 5 g of NaCl, 2.5 g of Na2HPO4 and 2 g of glucose for 1 L) 
	- Lysogeny Broth (LB) medium (10 g of peptone, 5 g of yeast extract and 10 g of NaCl for 1 L)</li>
	<li>For Northern blot assays, prepare the following buffers: 
	- Blocking solution (1x maleic acid and 1% blocking reagent) 
	- Hybridization solution (50% formamide, 5x SSC, 7% SDS, 1% blocking solution and 0.2% N-lauryl sarcosine, 50 mM sodium phosphate). Heat with agitation to dissolve. 
	CAUTION: Carefully follow the safety precautions related to each product. 
	- 1 M sodium phosphate (58 mM sodium phosphate dibasic and 42 mM sodium phosphate monobasic) 
	- Saline, sodium citrate (SSC) buffer, 20x concentrate (3 M NaCl and 300 mM trisodium citrate)</li>
</ol>
2. Safety issues 
<ol>
	<li>Carry out all steps involving viable pathogenic bacteria in a level 2 containment lab. 
	NOTE: Only cell extracts can be taken outside after lysis (step 5).</li>
	<li>Put on a lab coat and gloves.</li>
	<li>Ensure that the wrists are covered.</li>
	<li>Clean the biological safety cabinet (Class II) with a disinfectant solution.</li>
	<li>Dispose solid wastes exposed to bacteria in the appropriate biomedical bin.</li>
	<li>Treat flasks containing contaminated liquids with a disinfectant solution. Then, discard it in a sink.</li>
	<li>Carefully wash hands and wrists with soap and remove the lab coat before leaving the level 2 containment lab.</li>
</ol>
3. Plasmid construction 
NOTE:&#160;For cloning purposes, it is crucial to first identify the boundaries of the endogenous sRNA. The pCN51-P3 and pCN51-P3-MS2 plasmids&#160;are&#160;described in&#160;Tomasini et al. (2017)27.&#160;The P3 promoter allows high expression of the sRNA in a cell-density-dependent manner (i.e., when bacteria enter the stationary phase of growth). Many staphylococcal sRNAs accumulate during this growth phase.
<ol>
	<li>Amplify the sRNA sequence by PCR using a high-fidelity DNA polymerase and a PCR machine. Carefully follow manufacturer&#8217;s instructions and read Garibyan and Avashia (2013)31 for more details.</li>
	<li>Use the following templates to design the specific primers: 
	<img alt="Equation 1" class="xfigimg" src="/files/ftp_upload/61731/text12.png" /> 
	and 5&#8217;-CGCGGATCC(N)-3&#8217; for forward and reverse primers, respectively. 
	NOTE: These oligonucleotides enable to fuse the MS2 sequence (in bold) to the 5&#8217; end of the sRNA of interest. PstI and BamHI restriction sites (underlined) are added at the 5&#8217; and 3&#8217; extremities of the MS2-sRNA construct to clone the amplicon into the pCN51-P3 plasmid27. (N) corresponds to the gene-specific sequence&#160;(15-20 nucleotides).</li>
	<li>Digest 1 &#181;g of pCN51-P3 plasmid and 1 &#181;g of the MS2-sRNA PCR product with 2 U of PstI and 1 U of BamHI in the appropriate buffer according to manufacturer&#8217;s recommendations.</li>
	<li>Incubate 1 h at 37 &#176;C and purify DNA using a PCR purification kit (see Table of Materials).</li>
	<li>Mix the digested pCN51-P3 plasmid (300 ng) and MS2-sRNA amplicon (molar ratio for vector:insert = 1:3) in a 1.5 mL tube. See Revie et al. (1988)32 to maximize ligation efficiency. Add 1 &#181;L of the Ligase Buffer and 10 U of T4 Ligase in each tube. Adjust the volume to 10 &#181;L with ultrapure water.</li>
	<li>Incubate at 22 &#176;C for at least 2 h.</li>
	<li>Add 5 &#181;L of ligation mixture to 50 &#181;L&#160;of&#160;frozen DH5&#945;&#160;chemically competent E.&#160;coli cells. Read Seidman et al. (2001)33 to learn more about plasmid transformation and chemically competent cells.</li>
	<li>Incubate 30 min on ice.</li>
	<li>Heat shock (45 s at 42 &#176;C) the transformation tube using a heat block or water bath.</li>
	<li>Add 900 &#181;L of LB medium and incubate at 37 &#176;C for 30 min.</li>
	<li>Plate 100 &#181;L of the bacterial suspension on a LB agar plate supplemented with ampicillin (100 &#181;g/&#181;L). &#160; 
	NOTE: the pCN51-P3 vector encodes an ampicillin resistance gene, which enables to select only E. coli clones carrying the pCN51-P3-MS2-sRNA plasmid.</li>
	<li>Extract the pCN51-P3-MS2-sRNA plasmid from an overnight bacterial culture (5 mL) grown in the presence of ampicillin (100 &#181;g/&#181;L) using a plasmid DNA miniprep kit (see Table of Materials).</li>
	<li>Verify the construct by Sanger&#160;sequencing34 using the following primer, 5&#8217;-TCTCGAAAATAATAGAGGG-3&#8217;.&#160;</li>
	<li>Transform&#160;the pCN51-P3-MS2-sRNA plasmid into DC10B chemically competent E. coli cells.&#160;Repeat steps 3.7 to 3.11.</li>
	<li>Extract the pCN51-P3-MS2-sRNA plasmid (see step 3.12) and transform 1-5 &#181;g of plasmid DNA into HG001 &#916;sRNA electrocompetent S. aureus cells using an electroporation apparatus. Carefully follow manufacturer&#8217;s instructions. Read Grosser and Richardson (2016)35 to learn more about methods for preparing electrocompetent S. aureus. 
	CAUTION: This step involves handling of pathogenic bacteria (see step 2).</li>
	<li>Add 900 &#181;L of BHI medium and incubate at 37 &#176;C for 3 h.</li>
	<li>Centrifuge 1 min at 16,000 x g. Discard the supernatant.</li>
	<li>Resuspend the pellet in 100 &#181;L of BHI and plate the bacterial suspension on BHI agar plates supplemented with erythromycin (10 &#181;g/&#181;L). &#160; 
	NOTE: The pCN51-P3 vector also encodes an erythromycin resistance gene, which enables to select only S. aureus clones carrying the pCN51-P3-MS2-sRNA plasmid.</li>
</ol>
4. Bacteria harvesting
CAUTION: This step involves handling of pathogenic bacteria (see step 2).
<ol>
	<li>Grow one colony of strains carrying either pCN51-P3-MS2-sRNA or pCN51-P3-MS227 plasmids in 3 mL of BHI medium supplemented with erythromycin (10 &#181;g/&#181;L) in duplicates.</li>
	<li>Dilute each overnight culture in 50 mL (&#8776;1/100) of fresh BHI medium supplemented with erythromycin (10 &#181;g/&#181;L) to reach an OD600nm of 0.05. Use 250 mL sterilized flasks (5:1 flask-to-medium ratio). 
	NOTE: Medium and growth conditions should be set according to the expression pattern of the studied sRNA.</li>
	<li>Grow cultures at 37 &#176;C with shaking at 180 rpm for 6 h.</li>
	<li>Transfer each culture into a 50 mL centrifuge tube.</li>
	<li>Centrifuge at 2,900 x g during 15 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Keep pellets on ice and directly perform mechanical cell lysis (step 5) or freeze and store pellets at -80 &#176;C.</li>
</ol>
5. Mechanical cell lysis 
CAUTION: Following steps must be performed on ice and buffers must be at 4 &#176;C. Use gloves and take all precautions to protect samples from RNases.
<ol>
	<li>Resuspend pellets (step 4.6) in 5 mL of Buffer A.</li>
	<li>Transfer the resuspended cells in 15 mL centrifuge tubes with 3.5 g of silica beads (0.1 mm).</li>
	<li>Insert tubes in a mechanical cell lysis instrument (see Table of Materials). Run a cycle of 40 s at 4.0 m/s. 
	NOTE: If one cycle is not enough to break cells, let the device cool for 5 min while keeping samples on ice. Then, repeat another cycle of 40 s at 4.0 m/s. The efficiency of cell lysis can be tested by plating the supernatant on BHI-agar plate.</li>
	<li>Centrifuge at 15,700 x g for 15 min. Recover the supernatant and keep it on ice.</li>
</ol>
6. Column preparation
CAUTION: Be careful not to allow the amylose resin to dry. If needed, seal the column with an end-cap. Prepare all the solutions before starting the affinity purification.
<ol>
	<li>Put a chromatography column in a column rack (see Table of Materials).</li>
	<li>Remove the column tip and wash the column with ultrapure water.</li>
	<li>Add 300 &#181;L of amylose resin.</li>
	<li>Wash the column with 10 mL of Buffer A.</li>
	<li>Dilute 1,200 pmol of MBP-MS2 protein in 6 mL of Buffer A and load it into the column.</li>
	<li>Wash the column with 10 mL of Buffer A.</li>
</ol>
7. MS2-affinity purification (Figure 1)
<ol>
	<li>Load the cell lysate into the column. 
	NOTE: Keep 1 mL of the cell lysate (Crude extract, CE) to extract total RNA (step 8) and perform Northern blot (step 9) and transcriptomic (step 10) analysis.</li>
	<li>Collect the flow-through fraction (FT) in a clean collection tube.</li>
	<li>Wash the column 3 times with 10 mL of Buffer A. Collect the wash fraction (W).</li>
	<li>Elute the column with 1 mL of Buffer E and collect the elution fraction (E) in a 2 mL microtube.</li>
	<li>Keep all collected fractions on ice until RNA extraction (step 8) or freeze them at -20 &#176;C for later use.</li>
</ol>
8. RNA extraction of collected fractions (CE, FT, W and E)
<ol>
	<li>Use 1 mL of each fraction (including FT and W) for RNA extraction.</li>
	<li>Add 1 volume of phenol. Mix vigorously. 
	CAUTION: Phenol is volatile and corrosive, pay attention and work safely under a fume hood.</li>
	<li>Centrifuge at 16,000 x g for 10 min at 20 &#176;C.</li>
	<li>Transfer the upper phase in a clean 2 mL microtube.</li>
	<li>Add 1 volume of chloroform/isoamyl alcohol (24:1) and repeat steps 8.3 to 8.4. 
	CAUTION: Work safely under a fume hood.</li>
	<li>Add 2.5 volumes of cold ethanol 100% and 1/10 volume of 3 M sodium acetate (NaOAc) pH 5,2.</li>
	<li>Precipitate overnight at -20 &#176;C. 
	NOTE: Precipitation can also be performed in an ethanol/dry ice bath during 20 min or at -80 &#176;C during 2 h.</li>
	<li>Centrifuge at 16,000 x g for 15 min at 4 &#176;C. Slowly remove ethanol with a pipette while being careful not to disturb the pellet. 
	CAUTION: The RNA pellet is not always visible and is sometimes loose in presence of ethanol.</li>
	<li>Add 500 &#181;L of 80% cold ethanol.</li>
	<li>Centrifuge at 16,000 x g for 5 min at 4 &#176;C.</li>
	<li>Discard ethanol by pipetting it slowly. Dry the pellet using a vacuum concentrator, 5 min on run mode.</li>
	<li>Resuspend the pellet in an appropriate volume (15-50 &#181;L) of ultrapure water. Freeze the pellet at -20 &#176;C for later use.</li>
	<li>Assess RNA quantity (260 nm) and quality (260/280 and 260/230 wavelength ratios) using a spectrophotometer/fluorometer (see Table of Materials). Carefully follow manufacturer&#8217;s instructions. 
	NOTE: 3-4 &#181;g are generally obtained in the elution fraction (E). This mainly depends on tested conditions.</li>
</ol>
9. Analysis of MS2-affinity purification by Northern blot36
<ol>
	<li>Dilute 5 &#181;g of RNA of CE, FT, W fractions and 500 ng of E fraction in 10 &#181;L of ultrapure water and mix with 10 &#181;L of RNA loading buffer.</li>
	<li>Incubate 3 min at 90 &#176;C.</li>
	<li>Load samples into wells of an 1% agarose gel supplemented with 20 mM of guanidium thiocyanate and run the gel at 100-150 V in TBE 1x buffer at 4 &#176;C. Read Koontz (2013)37 for more details.</li>
	<li>Transfer RNAs on a nitrocellulose membrane by vacuum transfer for 1h or capillarity transfer overnight. 
	NOTE: The capillarity method is more efficient for large RNAs.</li>
	<li>UV cross-link RNAs on the membrane (120 mJ at 254 nm) using an ultraviolet crosslinker.</li>
	<li>Insert the membrane in a hybridization bottle with the RNA side facing up.</li>
	<li>Add 10-20 mL of preheated hybridization solution. Incubate 30 min at 68 &#176;C.</li>
	<li>Discard the solution and add 10-20 mL of fresh hybridization solution supplemented with 1 &#181;L of the sRNA-specific probe. Incubate overnight at 68 &#176;C. 
	NOTE: The DIG-labelled RNA probe is synthetized using a DIG RNA labelling kit and following manufacturer&#8217;s instructions. Alternatively, a radiolabelled probe can be used.</li>
	<li>Wash the membrane with 10-20 mL of wash solution 1 (2x SSC and 0.1% SDS) for 5 min at 20 &#176;C. Repeat once.</li>
	<li>Wash the membrane with 10-20 mL of wash solution 2 (0.2x SSC and 0.1% SDS) for 15 min at 68 &#176;C. Repeat once.</li>
	<li>Incubate with 10-20 mL of blocking solution for at least 30 min at 20 &#176;C.</li>
	<li>Discard the solution and add 10-20 mL of the blocking solution supplemented with the polyclonal anti-digoxigenin antibody (1/1000), conjugated to alkaline phosphatase. Incubate 30 min at 20 &#176;C.</li>
	<li>Wash the membrane with 10-20 mL of the wash solution 3 (1x maleic acid and 0.3% Tween 20) for 15 min at 20 &#176;C. Repeat once.</li>
	<li>Incubate the membrane with 10-20 mL of the detection solution (0.1 M Tris HCl and 0.1 M NaCl pH 9.5) 5 min at 20 &#176;C.</li>
	<li>Put the membrane on a plastic film and soak it with the substrate (see Table of Materials). Incubate 5 min in the dark.</li>
	<li>Seal the membrane in a plastic film. Put the membrane in an autoradiography cassette.</li>
	<li>Expose the membrane to an autoradiography film in the dedicated dark room. 
	NOTE: The exposition time depends on the signal strength, from few seconds to minutes.</li>
	<li>Reveal the exposed film in an automatic developing device.</li>
</ol>
10. Preparation of the samples for RNA sequencing 
NOTE: This step only concerns RNAs extracted from E and CE fractions.
<ol>
	<li>Add to each sample 10 &#181;L of 10x DNase buffer and DNase I (1 U/&#181;g of treated RNAs). Add water for a final volume of 100 &#181;L.</li>
	<li>Incubate 1 h at 37 &#176;C.</li>
	<li>Extract and purify RNAs as previously described (steps 8.2 to 8.11).</li>
	<li>Resuspend the RNA pellet in 20 &#181;L of ultrapure water. 
	NOTE: The presence of remaining DNA can be checked using PCR and specific primers (e.g., 16S gene).</li>
	<li>Assess RNA quantity and quality using a microfluidics-based electrophoresis analysis system (see Table of Materials). 
	NOTE: 1 &#181;g is generally obtained in the elution fraction (E) after DNase treatment.</li>
	<li>Remove ribosomal RNAs with a bacterial rRNA depletion kit. 
	NOTE: Large and abundant RNAs (i.e., rRNAs) tend to non-specifically interact with the affinity column. 500 ng of extracted RNA are required to perform this step.</li>
	<li>Again assess RNA quantity and quality using a microfluidics-based electrophoresis analysis system.</li>
	<li>Prepare cDNA libraries with 10-20 ng of ribodepleted RNA using a cDNA library preparation kit and following manufacturer&#8217;s instructions.</li>
	<li>Sequence the obtained libraries using a sequencing instrument (e.g., single-end, 150 bp; see Table of Materials). 
	NOTE: 5-10 million reads per sample are generally enough.</li>
</ol>
11. RNAseq data analysis (Figure 2)
<ol>
	<li>Download the FastQ sequencing files from the sequencing platform.</li>
	<li>Access to the Galaxy instance of Roscoff biological station (https://galaxy.sb-roscoff.fr/) and log in. 
	NOTE: Every mentioned algorithm can be easily found using the search bar. A user guide is provided for each tool. 
	CAUTION: The version of required tools may differ from the public Galaxy server38.</li>
	<li>Click on Get Data icon and then Upload File from your computer. Upload FastQ sequencing file of each MS2 control and MS2-sRNA samples. Upload also FASTA reference genome file and GFF annotation file.</li>
	<li>Run FastQC Read Quality reports (Galaxy Version 0.69). 
	NOTE: This tool provides a quality assessment of raw sequences (e.g., quality score, presence of adapter sequences).</li>
	<li>Run Trimmomatic flexible read trimming tool (Galaxy Version 0.36.6) to notably remove adapter sequences and poor-quality reads. Indicate adapter sequences used for library preparation (e.g., TruSeq 3, single-ended). Add the following Trimmomatic operations: SLIDINGWINDOW (Number of bases=4; Average quality=20) and MINLEN (Min length of reads=20).</li>
	<li>Run again FastQC Read Quality reports (Galaxy Version 0.69).</li>
	<li>Run Bowtie2 - map reads against reference genome (Galaxy Version 2.3.2.2). Use the Genome Reference FASTA file from the history to map reads with default settings (very sensitive local). 
	NOTE: BAM file generated by Bowtie2 tool can be visualized using the Integrative Genomics Viewer (IGV). Associated BAI file will also be required.</li>
	<li>Optionally, Run Flagstat which compiles stats for BAM dataset (Galaxy Version 2.0).</li>
	<li>Run htseq-count - Count (Galaxy Version 0.6.1) which aligns reads overlapping features in the GFF annotation file. Use the Intersection (non-empty) mode.</li>
	<li>Archive all raw counts files from htseq-count analysis into a single Zip file.</li>
	<li>Run SARTools DESeq2 to compare data (Galaxy Version 1.6.3.0). Provide the Zip file containing raw counts files and the design file, a tab delimited file describing the experiment. Carefully follow provided instructions to generate the design file.</li>
</ol>

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<li>Olejniczak, M., Storz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ProQ%2FFinO-domain+proteins%3A+another+ubiquitous+family+of+RNA+matchmakers%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers.</a> Molecular Microbiology. 104 (6), 905-915 (2017).</li>
<li>Lalaouna, D., Desgranges, E., Caldelari, I., Marzi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+Sixteen+-+MS2-Affinity+Purification+Coupled+With+RNA+Sequencing+Approach+in+the+Human+Pathogen+Staphylococcus+aureus%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Chapter Sixteen - MS2-Affinity Purification Coupled With RNA Sequencing Approach in the Human Pathogen Staphylococcus aureus.</a> Methods in Enzymology. 612, 393-411 (2018).</li>
<li>Lalaouna, D., Prevost, K., Eyraud, A., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+unknown+RNA+partners+using+MAPS%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Identification of unknown RNA partners using MAPS.</a> Methods. 117, 28-34 (2017).</li>
<li>Lalaouna, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+3%27+external+transcribed+spacer+in+a+tRNA+transcript+acts+as+a+sponge+for+small+RNAs+to+prevent+transcriptional+noise%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">A 3' external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent transcriptional noise.</a> Molecular Cell. 58 (3), 393-405 (2015).</li>
<li>Lalaouna, D., Morissette, A., Carrier, M. C., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DsrA+regulatory+RNA+represses+both+hns+and+rbsD+mRNAs+through+distinct+mechanisms+in+Escherichia+coli%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.</a> Molecular Microbiology. 98 (2), 357-369 (2015).</li>
<li>Lalaouna, D., Prevost, K., Laliberte, G., Houe, V., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contrasting+silencing+mechanisms+of+the+same+target+mRNA+by+two+regulatory+RNAs+in+Escherichia+coli%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli.</a> Nucleic Acids Research. 46 (5), 2600-2612 (2018).</li>
<li>Lalaouna, D., Eyraud, A., Devinck, A., Prevost, K., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GcvB+small+RNA+uses+two+distinct+seed+regions+to+regulate+an+extensive+targetome%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">GcvB small RNA uses two distinct seed regions to regulate an extensive targetome.</a> Molecular Microbiology. 111 (2), 473-486 (2019).</li>
<li>Silva, I. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SraL+sRNA+interaction+regulates+the+terminator+by+preventing+premature+transcription+termination+of+rho+mRNA%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA.</a> Proceedings of the National Academy of Sciences. 116 (8), 3042-3051 (2019).</li>
<li>Lalaouna, D., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+sRNA+interacting+with+a+transcript+of+interest+using+MS2-affinity+purification+coupled+with+RNA+sequencing+%28MAPS%29+technology%5BTitle%5D)+AND+%22Genomics+Data%22%5BJournal%5D)">Identification of sRNA interacting with a transcript of interest using MS2-affinity purification coupled with RNA sequencing (MAPS) technology.</a> Genomics Data. 5, 136-138 (2015).</li>
<li>Tomasini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA+targetome+of+Staphylococcus+aureus+non-coding+RNA+RsaA%3A+impact+on+cell+surface+properties+and+defense+mechanisms%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms.</a> Nucleic Acids Research. 45 (11), 6746-6760 (2017).</li>
<li>Bronesky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+multifaceted+small+RNA+modulates+gene+expression+upon+glucose+limitation+in+Staphylococcus+aureus%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus.</a> The EMBO Journal. 38 (6), (2019).</li>
<li>Lalaouna, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RsaC+sRNA+modulates+the+oxidative+stress+response+of+Staphylococcus+aureus+during+manganese+starvation%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation.</a> Nucleic Acids Research. 47 (1), 9871-9887 (2019).</li>
<li>Carrier, M. C., Laliberte, G., Masse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+New+Bacterial+Small+RNA+Targets+Using+MS2+Affinity+Purification+Coupled+to+RNA+Sequencing%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Identification of New Bacterial Small RNA Targets Using MS2 Affinity Purification Coupled to RNA Sequencing.</a> Methods in Molecular Biology. 1737, 77-88 (2018).</li>
<li>Garibyan, L., Avashia, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymerase+chain+reaction%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Polymerase chain reaction.</a> Journal of Investigative Dermatology. 133 (3), 1-4 (2013).</li>
<li>Revie, D., Smith, D. W., Yee, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+analysis+for+optimization+of+DNA+ligation+reactions%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Kinetic analysis for optimization of DNA ligation reactions.</a> Nucleic Acids Research. 16 (21), 10301-10321 (1988).</li>
<li>Seidman, C. E., Struhl, K., Sheen, J., Jessen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introduction+of+plasmid+DNA+into+cells%5BTitle%5D)+AND+%22Current+Protocols+in+Molecular+Biology%22%5BJournal%5D)">Introduction of plasmid DNA into cells.</a> Current Protocols in Molecular Biology. , Chapter 1 Unit 1.8 (2001).</li>
<li>Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., Roe, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cloning+in+single-stranded+bacteriophage+as+an+aid+to+rapid+DNA+sequencing%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing.</a> Journal of Molecular Biology. 143 (2), 161-178 (1980).</li>
<li>Grosser, M. R., Richardson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+Preparation+and+Electroporation+of+S.+aureus+and+S.+epidermidis%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Method for Preparation and Electroporation of S. aureus and S. epidermidis.</a> Methods in Molecular Biology. 1373, 51-57 (2016).</li>
<li>Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Northern+blot+analysis%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Northern blot analysis.</a> Methods in Molecular Biology. 58, 113-128 (1996).</li>
<li>Koontz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agarose+gel+electrophoresis%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Agarose gel electrophoresis.</a> Methods in Enzymology. 529, 35-45 (2013).</li>
<li>Afgan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Galaxy+platform+for+accessible%2C+reproducible+and+collaborative+biomedical+analyses%3A+2016+update%5BTitle%5D)+AND+%22Nucleic+Acids+Reseaerch%22%5BJournal%5D)">The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update.</a> Nucleic Acids Reseaerch. 44, 3-10 (2016).</li>
<li>Jagodnik, J., Brosse, A., Le Lam, T. N., Chiaruttini, C., Guillier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanistic+study+of+base-pairing+small+regulatory+RNAs+in+bacteria%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Mechanistic study of base-pairing small regulatory RNAs in bacteria.</a> Methods. 117, 67-76 (2017).</li>
<li>Mann, M., Wright, P. R., Backofen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IntaRNA+2.0%3A+enhanced+and+customizable+prediction+of+RNA-RNA+interactions%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions.</a> Nucleic Acids Res. 45, 435-439 (2017).</li>
<li>Georg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+power+of+cooperation%3A+Experimental+and+computational+approaches+in+the+functional+characterization+of+bacterial+sRNAs%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">The power of cooperation: Experimental and computational approaches in the functional characterization of bacterial sRNAs.</a> Molecular Microbiology. 113 (3), 603-612 (2020).</li>
</ol>

The authors have nothing to disclose.

A modified protocol for Gram-positive bacteria 
The initial protocol of MAPS was developed to study sRNA interactome in the model organism E. coli20,30. Here, we describe a modified protocol which is suitable for the characterization of sRNA-dependent regulatory networks in the opportunistic human pathogen S. aureus and is certainly transposable to other Gram-positive bacteria, pathogenic or not.
<p class="jove_content"...

Hundreds, perhaps even thousands of small regulatory RNAs (sRNAs) have been identified in most bacterial genomes, but the functions of the vast majority of them remain uncharacterized. Overall, sRNAs are short non-coding molecules, playing major roles in bacterial physiology and adaptation to fluctuating environments1,2,3. Indeed, these macromolecules are at the center of numerous intricate regulatory networks, impacting metabolic pathways, stress responses but also virulence and antibiotic resistance. Logically, their synthesis is triggered by specific environment stimuli (e.g., nutrient starvation, oxidative or membrane stresses). Most sRNAs regulate multiple target mRNAs at the post-transcriptional level through short and non-contiguous base pairing. They usually prevent translation initiation by competing with ribosomes for translation initiation regions4. The formation of sRNA:mRNA duplexes also often results in the active degradation of the target mRNA by recruitment of specific RNases.
The characterization of an sRNA targetome (i.e., the whole set of its target RNAs) allows the identification of the metabolic pathways in which it intervenes and the potential signal it answers to. Consequently, the functions of a specific sRNA can generally be inferred from its targetome. For this purpose, several in silico prediction tools have been developed such as IntaRNA and CopraRNA5,6,7. They notably rely on sequence complementarity, pairing energy and accessibility of the potential interaction site to determine putative sRNA partners. However, prediction algorithms do not integrate all factors influencing base-pairing in vivo such as the involvement of RNA chaperones8 favoring sub-optimal interactions or the co-expression of both partners. Due to their inherent limitations, the false positive rate of prediction tools remains high. Most experimental large-scale approaches are based on the co-purification of sRNA:mRNA couples interacting with a tagged RNA binding protein (RBP)6,9. For example, the RNA Interaction by Ligation and sequencing (RIL-seq) method identified RNA duplexes co-purified with RNA chaperones such as Hfq and ProQ in Escherichia coli10,11. A similar technology called UV-Crosslinking, Ligation And Sequencing of Hybrids (CLASH) was applied to RNase E- and Hfq-associated sRNAs in E. coli12,13. Despite the well-described roles of Hfq and ProQ in sRNA-mediated regulation in multiple bacteria8,14,15, sRNA-based regulation seems to be RNA chaperone-independent in several organisms like S. aureus16,17,18. Even if the purification of RNA duplexes in association with RNases is feasible as demonstrated by Waters and coworkers13, this remains tricky as RNases trigger their rapid degradation. Hence, the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) approach19,20 constitutes a solid alternative in such organisms.
Unlike above-mentioned methods, MAPS uses a specific sRNA as bait to capture all interacting RNAs and hence does not rely on the involvement of an RBP. The entire process is depicted in Figure 1. In brief, the sRNA is tagged at the 5&#8217; with the MS2 RNA aptamer that is specifically recognized by the MS2 coat protein. This protein is fused with the maltose binding protein (MBP) to be immobilized on an amylose resin. Therefore, MS2-sRNA and its RNA partners are retained on the affinity chromatography column. After elution with maltose, co-purified RNAs are identified using high-throughput RNA sequencing followed by bioinformatic analysis (Figure 2). The MAPS technology ultimately draws an interacting map of all potential interactions occurring in vivo.
MAPS technology was originally implemented in the non-pathogenic Gram-negative bacterium E. coli21. Remarkably, MAPS helped identify a tRNA-derived fragment specifically interacting with both RyhB and RybB sRNAs and preventing any sRNA transcriptional noise to regulate mRNA targets in non-inducing conditions. Thereafter, MAPS has been successfully applied to other E. coli sRNAs like DsrA22, RprA23, CyaR23 and GcvB24 (Table 1). In addition to confirming previously known targets, MAPS extended the targetome of these well-known sRNAs. Recently, MAPS has been performed in Salmonella Typhimurium and revealed that SraL sRNA binds to rho mRNA, coding for a transcription termination factor25. Through this pairing, SraL protects rho mRNA from the premature transcription termination triggered by Rho itself. Interestingly, this technology is not restricted to sRNAs and can be applied to any type of cellular RNAs as exemplified by the use of a tRNA-derived fragment26 and a 5&#8217;-untranslated region of mRNA22 (Table 1).
MAPS method has been also adapted to the pathogenic Gram-positive bacterium S. aureus19. Specifically, the lysis protocol has been widely modified to efficiently break cells due to a thicker cell wall than Gram-negative bacteria and to maintain RNA integrity. This adapted protocol already unravelled the interactome of RsaA27, RsaI28 and RsaC29. This approach gave insights into the crucial role of these sRNAs in regulatory mechanisms of cell surface properties, glucose uptake, and oxidative stress responses.
The protocol developed and implemented in E. coli in 2015 has been recently described in great detail30. Here, we provide the modified MAPS protocol, which is particularly suitable for studying sRNA regulatory networks in Gram-positive (thicker cell wall) bacteria whether non-pathogenic or pathogenic (safety precautions).

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>2 mL microcentrifuge tube</td><td>Starstedt</td><td>72.691</td><td></td></tr><tr><td>2100 Bioanalyzer Instrument</td><td>Agilent</td><td>G2939BA</td><td>RNA quantity and quality</td></tr><tr><td>250 mL culture flask</td><td>Dominique Dutscher</td><td>2515074</td><td>Bacterial cultures</td></tr><tr><td>50 mL centrifuge tubes</td><td>Falcon</td><td>352051</td><td>Culture centrifugation</td></tr><tr><td>Absolute ethanol</td><td>VWR Chemicals</td><td>20821.321</td><td>RNA extraction and purification</td></tr><tr><td>Allegra X-12R Centrifuge</td><td>Beckman Coulter</td><td></td><td>Bacterial pelleting</td></tr><tr><td>Ampicilin (amp)</td><td>Sigma-Aldrich</td><td>A9518-5G</td><td>Growth medium</td></tr><tr><td>Amylose resin</td><td>New England BioLabs</td><td>E8021S</td><td>MS2-affinity purification</td></tr><tr><td>Anti-dioxigenin AP Fab fragment</td><td>Sigma Aldrich</td><td>11093274910</td><td>Northern blot assays</td></tr><tr><td>Autoradiography cassette</td><td>ThermoFisher Scientific</td><td>50-212-726</td><td>Northern blot assays</td></tr><tr><td>BamHI</td><td>ThermoFisher Scientific</td><td>ER0051</td><td>Plasmid construction</td></tr><tr><td>BHI (Brain Heart Infusion) Broth</td><td>Sigma-Aldrich</td><td>53286</td><td>Growth medium</td></tr><tr><td>Blocking reagent</td><td>Sigma Aldrich</td><td>11096176001</td><td>Northern blot assays</td></tr><tr><td>CDP-Star</td><td>Sigma Aldrich</td><td>11759051001</td><td>Northern blot assays (substrate)</td></tr><tr><td>Centrifuge 5415 R</td><td>Eppendorf</td><td></td><td>RNA extraction and purification</td></tr><tr><td>Chloroform</td><td>Dominique Dutscher</td><td>508320-CER</td><td>RNA extraction and purification</td></tr><tr><td>DIG-RNA labelling mix</td><td>Sigma-Aldrich</td><td>11277073910</td><td>Northern blot assays</td></tr><tr><td>DNase I</td><td>Roche</td><td>4716728001</td><td>DNase treatment</td></tr><tr><td>Erythromycin (ery)</td><td>Sigma-Aldrich</td><td>Fluka 45673</td><td>Growth medium</td></tr><tr><td>FastPrep device</td><td>MP Biomedicals</td><td>116004500</td><td>Mechanical lysis</td></tr><tr><td>Guanidium Thiocyanate</td><td>Sigma-Aldrich</td><td>G9277-250G</td><td>Northern blot assays</td></tr><tr><td>Hybridization Hoven Hybrigene</td><td>Techne</td><td>FHB4DD</td><td>Northern blot assays</td></tr><tr><td>Hybridization tubes</td><td>Techne</td><td>FHB16</td><td>Northern blot assays</td></tr><tr><td>Isoamyl alcohol</td><td>Fisher Scientific</td><td>A/6960/08</td><td>RNA extraction and purification</td></tr><tr><td>LB (Lysogeny Broth)</td><td>Sigma-Aldrich</td><td>L3022</td><td>Growth medium</td></tr><tr><td>Lysing Matrix B Bulk</td><td>MP Biomedicals</td><td>6540-428</td><td>Mechanical lysis</td></tr><tr><td>MicroPulser Electroporator</td><td>BioRad</td><td>1652100</td><td>Plasmid construction</td></tr><tr><td>Milli-Q water device</td><td>Millipore</td><td>Z00QSV0WW</td><td>Ultrapure water</td></tr><tr><td>NanoDrop spectrophotometer</td><td>ThermoFisher Scientific</td><td></td><td>RNA/DNA quantity and quality</td></tr><tr><td>Nitrocellulose membrane</td><td>Dominique Dutsher</td><td>10600002</td><td>Northern blot assays</td></tr><tr><td>Phembact Neutre</td><td>PHEM Technologies</td><td>BAC03-5-11205</td><td>Cleaning and decontamination</td></tr><tr><td>Phenol</td><td>Carl Roth</td><td>38.2</td><td>RNA extraction and purification</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>New England Biolabs</td><td>M0530</td><td>Plasmid construction</td></tr><tr><td>pMBP-MS2</td><td>Addgene</td><td>65104</td><td>MS2-MBP production</td></tr><tr><td>Poly-Prep chromatography column</td><td>BioRad</td><td>7311550</td><td>MS2-affinity purification</td></tr><tr><td>PstI</td><td>ThermoFisher Scientific</td><td>ER0615</td><td>Plasmid construction</td></tr><tr><td>Qubit 3 Fluorometer</td><td>Invitrogen</td><td>15387293</td><td>RNA quantity</td></tr><tr><td>RNAPro Solution</td><td>MP Biomedicals</td><td>6055050</td><td>Mechanical lysis</td></tr><tr><td>ScriptSeq Complete Kit</td><td>Illumina</td><td>BB1224</td><td>Preparation of cDNA librairies</td></tr><tr><td>Spectrophotometer Genesys 20</td><td>ThermoFisher Scientific</td><td>11972278</td><td>Bacterial cultures</td></tr><tr><td>SpeedVac Savant vacuum device</td><td>ThermoFisher Scientific</td><td>DNA120</td><td>RNA extraction and purification</td></tr><tr><td>Stratalinker UV Crosslinker 1800</td><td>Stratagene</td><td>400672</td><td>Northern blot assays</td></tr><tr><td>T4 DNA ligase</td><td>ThermoFisher Scientific</td><td>EL0014</td><td>Plasmid construction</td></tr><tr><td>TBE (Tris-Borate-EDTA)</td><td>Euromedex</td><td>ET020-C</td><td>Northern blot assays</td></tr><tr><td>ThermalCycler T100</td><td>BioRad</td><td>1861096</td><td>Plasmid construction</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P9416-100ML</td><td>Northern blot assays</td></tr><tr><td>X-ray film processor</td><td>hu.q</td><td>HQ-350XT</td><td>Northern blot assays</td></tr><tr><td>X-ray films Super RX-N</td><td>FujiFilm</td><td>4741019318</td><td>Northern blot assays</td></tr></tbody></table>

ms2-affinity purification coupled with rna sequencing in gram-positive bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

The representative results originate from the study of RsaC targetome in S. aureus29. RsaC is an unconventional 1,116 nt-long sRNA. Its 5&#8217; end contains several repeated regions while its 3&#8217; end (544 nt) is structurally independent and contains all predicted interaction sites with its mRNA targets. The expression of this sRNA is induced when manganese (Mn) is scarce, which is often encountered in the context of host immune response. Using MAPS technology, we identified several ...

Watch this Scientific Journal Video about MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria at JoVE.com

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

ms2-affinity-purification-coupled-with-rna-sequencing-gram-positive

Research

JoVE Journal

Biochemistry

6.6K Views.  Université de Strasbourg, CNRS. MS2-affiniteitszuivering in combinatie met RNA-sequencing of MAPS is ontwikkeld om het hele doel van elk RNA dat van belang is voor bacteriën te identificeren. Deze technologie maakt het mogelijk om elke vorm van sRNA-partners onafhankelijk van het reguleringsmechanisme te identificeren. De karakterisering van sRNA-afhankelijke regulerende netwerken zal helpen om betere virulentiefactoren productie, biofilmvorming en overleving van de bacteriën in de gastheercellen te begrijpen en uiteindel...

Video: MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Genetics

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Immunology and Infection

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Biology

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

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