RNA extraction was performed as previously described (11 (link)). DNA probes for RNA detection are listed in Supplementary Table S3 . We separated 10 μg each of total RNAs onto 8% polyacrylamide/8M urea gels and transferred them onto Hybond-N+ membranes (Amersham). Specific 32P-labeled probes were hybridized in ExpressHyb solution (Clontech), then washed, exposed, and scanned with a PhosphorImager (Molecular Dynamics). Quantifications were performed with ImageQuant and normalized to either transfer-messenger RNA (tmRNA) or 5S ribosomal RNA. For the RNA half-life measurements, S. aureus HG003 was cultured overnight, diluted to 1/100, grown for an additional 3 h at 37°C to the exponential growth phase, and finally incubated with 200 μg/ml rifampicin for 1–120 min. Samples of 2 ml each were taken before and then at 1, 3, 5, 7.5, 10, 15, 20, 30, 60 and 120 min after rifampicin addition. These samples were centrifuged, and pellets were frozen in liquid nitrogen then stored at −80°C. The RNA was then extracted and northern blot assays performed.
Expresshyb solution
Manufactured by Takara Bio
Sourced in United States
ExpressHyb solution is a rapid hybridization buffer designed for use in Northern and Southern blotting applications. It facilitates efficient and rapid hybridization of labeled nucleic acid probes to target sequences on membranes.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (6 mentions)
Zeta probe gt membrane, by Bio-Rad (4 mentions)
Phosphorimager, by GE Healthcare (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Hybond n membrane, by Cytiva (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Typhoon fla 9500 scanner, by GE Healthcare (2 mentions)
X ray film, by Kodak (2 mentions)
Urea 10 polyacrylamide tbe gel, by Thermo Fisher Scientific (2 mentions)
Dynamarker small rna plus, by Thermo Fisher Scientific (2 mentions)
Comparative cycle threshold method, by Thermo Fisher Scientific (1 mentions)
Fastprep apparatus, by MP Biomedicals (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Blocking buffer, by Thermo Fisher Scientific (1 mentions)
Rna loading buffer, by Takara Bio (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
North2south biotin random prime dna labeling kit, by Thermo Fisher Scientific (1 mentions)
Versadoc imaging system, by Bio-Rad (1 mentions)
Dig dna labeling and detection kit, by Roche (1 mentions)
16 protocols using expresshyb solution
1
RNA Extraction and Half-life Analysis
2
Quantitative Analysis of S. aureus sRNAs
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S. aureus strains were grown in Luria-Bertani medium and then harvested. Cells were isolated by centrifugation and dissolved in a solution of 33 mmol/L sodium acetate, 17 mmol/L sodium dodecyl sulfate, and 1 mmol/L EDTA (pH 5.5). The cells were then mixed with glass beads and lysed by using a Fast Prep Apparatus (MP Biochemicals, LLC, Santa Ana, CA, USA).
RNAs were isolated by using water-saturated phenol (pH 5.0). RNAs were precipitated and washed with ethanol. Northern blotting of RNA markers was conducted by loading 10 μg of total RNA onto 8 mol/L urea, 8% polyacrylamide gels. Gels were subjected to electrophoresis and blotted onto nylon membranes at 30 V for 1.5 h in 0.5× Tris-HCl, borate, EDTA buffer.
Prehybridization and hybridization were performed by using ExpressHyb solution (Clontech, Mountain View, CA, USA) and 32P-labeled DNAs (Technical Appendix Table 2). Signals were detected by using phosphorimaging (Biocompare, South San Francisco, CA, USA) and quantified. Expression levels of sRNAs in strains were monitored by using quantitative PCR and specific primers (Technical Appendix Table 3). cDNAs were produced by using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Using the comparative cycle threshold method (Applied Biosystems), we normalized sRNA counts against transfer–messenger RNA (tmRNA) and S. aureus reference strain L102.
RNAs were isolated by using water-saturated phenol (pH 5.0). RNAs were precipitated and washed with ethanol. Northern blotting of RNA markers was conducted by loading 10 μg of total RNA onto 8 mol/L urea, 8% polyacrylamide gels. Gels were subjected to electrophoresis and blotted onto nylon membranes at 30 V for 1.5 h in 0.5× Tris-HCl, borate, EDTA buffer.
Prehybridization and hybridization were performed by using ExpressHyb solution (Clontech, Mountain View, CA, USA) and 32P-labeled DNAs (
3
Quantitative Analysis of RNA Expression
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Total RNA were prepared as previously described (15 (link)). For SprD, SprX2 and RNAIII, northern blots were done using 10 μg total RNA as we have previously described (10 (link)). Specific 32P-labeled probes (the sequences are in Supplementary Table S2 ) were hybridized with membranes in ExpressHyb solution (Clontech) for 90 min at 37°C, washed, exposed, then scanned with a Typhoon FLA 9500 scanner (GE Healthcare). For the quantitative real-time PCR (qRT-PCR), cDNAs were prepared using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR experiments were performed using Power SYBR Green PCR Master Mix (Applied Biosystems) with the primers listed in Supplementary Table S2 . Three independent experiments were performed, with independent RNA purifications. The hu gene was used for normalization.
4
Northern Blotting Protocol for RNA Analysis
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Briefly, RNA for Northern blotting (NB) was extracted using TRIzol (Life Technologies) according to the manufacturer’s instructions. RNA (20 μg) was mixed with 2×RNA loading buffer (TAKARA) and EB, denatured at 65°C for 15 minutes and then run on a 1.2% agarose/formaldehyde gel and transferred by capillary action onto a nitrocellulose membrane (Millipore). The nitrocellulose membrane was prehybridized with ExpressHyb solution (Clontech) at 42°C for 2 hours. Probes were produced using a North2South Biotin Random Prime DNA Labeling Kit (Thermo Scientific). Primers used for the production of probes are shown in Table 1 . The membrane was hybridized at 42°C overnight with fresh solution containing the corresponding probe and then washed twice at room temperature for 30 min with wash solution 3 (2×SSC, 0.1% SDS) and once at 42°C for 30 min with wash solution 2 (0.1×SSC, 0.1% SDS). The membrane was then blocked with blocking buffer (catalogue #89880A; Thermo Scientific) at room temperature for 30 min. Finally, the membrane was incubated with IRDye 800-conjugated streptavidin diluted in TBST (1:2500) and imaged on an Odyssey CLx infrared imaging system (Li-COR Biosciences).
5
Telomere Length Quantification by Blotting
6
Northern Blot Analysis of SprX and yabJ-spoVG
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Total RNAs were prepared as previously described (20 (link)). For SprX, northern blots were performed with 10 µg of total RNA (15 (link)). Specific 32P-labeled probes (Supplementary Table S2 ) were hybridized with ExpressHyb solution (Clontech) for 90 min, washed, exposed and then scanned with a Typhoon FLA 9500 scanner (GE Healthcare). For the northern blots of yabJ-spoVG mRNA, 10 µg of total RNAs were separated by 5% polyacrylamide/8M urea gel. The primer pair oSTM29/oSTM30 (Supplementary Table S2 ) was used to generate α32P-dCTP-labeled spoVG-specific probes by PCR labeling. For in vitro experiments, RNAs were transcribed from PCR fragments generated from genomic DNA using the primers described in Supplementary Table S2 . To produce the template-encoding SprX_mutL3, mutagenized oligonucleotides were used (Supplementary Table S2 ). RNA was produced by in vitro transcription using MEGAscript (Ambion). 5′-RNA γ32P-dATP labeling was performed (34 (link)). The RNA was purified by 8% polyacrylamide/8M urea gel, eluted, ethanol precipitated, then stored at −80°C.
7
RNA Extraction and Northern Blot Analysis
8
Transcriptional Analysis of Bacterial Growth
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RNA extractions were performed at three time points during growth – middle exponential (ME, OD 0.7), late exponential (LE, OD 1.6), and early stationary (ES, OD 1.9). Extractions were performed as reported31 (link). Cell pellets were dissolved into 500 µL of lysis buffer, and cells were broken by acid-treated glass beads and phenol. Total RNA was extracted by phenol/chloroform and ethanol precipitated overnight. RNA samples (15 μg) were loaded on denaturing 7.5% PAGE and transferred onto Zeta probe GT membranes (Bio-Rad) in 0.5 × TBE. Membranes were hybridized with specific 32P-labeled probes in ExpressHyb solution (Clontech, USA), washed, exposed, and scanned onto a PhosphorImager (Molecular Dynamics). For the RT-qPCR experiments, cDNA synthesis was performed with the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) and quantitative PCR with the Power Sybr® Green PCR Master Mix (Thermofisher Scientific). Transcript levels were determined by the ΔΔCt method, with adk as the control (Supplementary Table S5 ). Each experiment was performed in triplicates.
9
MICA and MICB mRNA Expression Analysis
10
RNA Expression Analysis by Northern Blot
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Total RNA was isolated using an RNeasy Kit (Qiagen, Valencia, CA). Fifteen-microgram aliquots of total RNA were size-fractionated in a 1% denaturing agarose-formaldehyde gel, transferred onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ), and cross-linked with ultraviolet radiation (Stratalinker; Stratagene). Hybridizations were performed in ExpressHyb solution (Clontech) at 65°C for 2 h with full-length rat preproinsulin cDNA labeled with 32P-dCTP by random primer labeling (Amersham Pharmacia Biotech). After hybridization, membranes were washed at 65°C in 0.1× SSC buffer containing 0.1% SDS. The results were visualized using a Phosphor Imager (Fuji BAS 1500; Fuji, Tokyo, Japan). Northern blots were stripped and re-probed with GAPDH cDNA to control for RNA loading.
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