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Supporting information
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Ultra-high-sensitivity stable-isotope probing of rRNA by high-throughput
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sequencing of isopycnic centrifugation gradients
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Tomo Aoyagi, Satoshi Hanada, Hideomi Itoh, Yuya Sato, Atsushi Ogata,
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Michael W. Friedrich, Yoshitomo Kikuchi, and Tomoyuki Hori
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Supplementary Experimental Procedures
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Preparation of RNA standard solutions
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Escherichia coli K12 (ATCC 10798) and Bacillus subtilis 168 (BGSC 1A700) were
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used to prepare the RNA standard solutions. Three types of RNA solution were
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prepared: (i) 13C-labeled RNA from E. coli, (ii) unlabeled RNA from E. coli, and (iii)
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unlabeled RNA from B. subtilis. Briefly, 13C-labeled RNA was extracted from the E.
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coli cells grown with [U-13C6]glucose (99 atom%; Sigma-Aldrich) as a sole carbon and
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energy source (Kindler et al., 2006). Unlabeled RNAs were extracted from cells of E.
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coli and B. subtilis, both of which were cultured with unlabeled glucose. The basal
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media (pH 7.2) for cultivation consisted of (in grams per liter) Na2HPO4, 6.97; KH2PO4,
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3.42; MgCl2•6H2O, 0.18; NH4Cl, 1.0; CaCl2•2H2O, 0.13; FeSO4•7H2O, 0.018;
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ZnSO4•7H2O, 0.00035; MnSO4•5H2O, 0.00031; and glucose, 2 (Kindler et al., 2006).
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Total nucleic acids were extracted using a direct lysis protocol involving bead beating as
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described previously (Noll et al., 2005). Then, RNA was purified by DNA digestion
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with the RQ1 DNase (Promega). Total RNA was quantified using a RiboGreen RNA
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quantification kit (Life Technologies) according to the manufacturer’s instructions.
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Mixing of RNA standard solutions and subsequent ultracentrifugation
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of 1%, 0.5%, 0.05%, 0.01%, 0.001%, and 0.0001%, and the resulting solution was
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designated the 13C mixture. Thus a ratio of 1% means a 1:99 mixture of 13C-labeled
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RNA and unlabeled RNA, respectively. In place of 13C-labeled RNA, unlabeled RNA
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from E. coli was mixed with that from B. subtilis at the same ratios, and this was
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designated the unlabeled mixture. Schematic overviews of the RNA mixtures used and
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examinations conducted are shown in Fig. S1. Mixtures (500 ng total RNA content
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each) of RNAs from E. coli and B. subtilis were added to the cesium trifluoroacetate
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(CsTFA) solution (Wako), formamide, and a gradient buffer (Tris, 0.1 M; KCL, 0.1 M;
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and EDTA, 1 mM), followed by density separation of RNAs by ultracentrifugation with
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128,000 g for >60 h at 20ºC (Lueders et al., 2004). Isopycnic centrifugation of the 13C
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mixture and the unlabeled mixture at ratios of 1%, 0.5%, 0.05%, and 0.01% was
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performed in duplicate for T-RFLP, while the centrifugation of the mixtures at ratios of
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0.05%, 0.01%, 0.001%, and 0.0001% was performed in triplicate for Illumina
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sequencing and qRT-PCR. Isopycnic centrifugation gradients were fractionated and then
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retrieved and purified by isopropanol precipitation. The CsTFA BD of each density
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fraction was determined with a refractometer (AR200; Reichert) (Lueders et al., 2004).
C-labeled RNA from E. coli was mixed with unlabeled RNA from B. subtilis at ratios
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T-RFLP analysis of the density-separated RNAs
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RNA from each density fraction from the 13C mixture and the unlabeled mixture was
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subjected to RT-PCR using a one-step RT-PCR system (Access Quick; Promega) with
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the primer set B27f/B907r (Hori et al., 2007), and the forward primer was labeled with
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a fluorescent dye (Beckmann Dye D4; Sigma-Aldrich). The thermal conditions of
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RT-PCR were the same as described previously (Hori et al., 2007), except that a total of
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16 cycles were employed for the PCR amplification. Density fractions of RNA with
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BDs of 1.750–1.806 g ml–1 were successfully amplified. The absence of DNA
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contamination was confirmed by RT-PCR without reverse transcriptase. RT-PCR
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amplicons were purified using a QIAquick PCR purification kit (QIAGEN). Two
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hundred ng of the amplicon was digested with an MspI restriction enzyme (New
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England Biolabs). The digest was mixed with a loading solution (Beckman Coulter)
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containing an internal size standard (Standard-600 kit; Beckman Coulter). T-RFs were
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size-separated by capillary array electrophoresis with a GenomLab GeXP (Beckman
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Coulter) and analyzed by peak height with a CEQ8000 Genetic Analysis system
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(Beckman Coulter).
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Illumina sequencing analysis of the selected density fractions of RNA
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The heaviest (“1H”), second-heaviest (“2H”), and light (“L”) fractions of RNA with
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BDs of 1.797–1.798 g ml–1, 1.790–1.792 g ml–1, and 1.765–1.776 g ml–1, respectively,
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were subjected to RT-PCR with a one-step RT-PCR system (Access Quick; Promega).
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The primers used for Illumina sequencing were 515f and 806r; both primers were
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modified to contain an Illumina adapter region, and the reverse primer was encoded
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with 12-bp barcodes (Caporaso et al., 2012). Each of the forward and reverse primers
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was at least 60 bp in length. The thermal conditions of RT-PCR were described
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previously (Hori et al., 2007), except that a total of 25 cycles and an annealing
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temperature of 54°C were employed for the PCR amplification. The amplification
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efficiency of RT-PCR with the long primers for Illumina sequencing was lower than that
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with common short PCR primers. Therefore, more amplification cycles were employed
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in RT-PCR for Illumina sequencing in order to yield a density of amplicons equivalent
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to the density that was successfully amplified by RT-PCR for T-RFLP. Agarose gel
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electrophoresis was used to confirm that the amount of RT-PCR product in each density
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fraction was almost the same between Illumina sequencing and T-RFLP. The RT-PCR
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amplicon was purified first with an AMPure XP Kit (Beckman Coulter) and then with a
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QIAquick gel extraction kit (QIAGEN). The DNA concentration of the purified
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amplicon was determined spectrophotometrically with a Quant-iT PicoGreen dsDNA
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reagent and kit (Life Technologies). An appropriate amount of the purified amplicon
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(i.e., the barcode-encoded library) and an internal control (PhiX Control V3; Illumina)
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was subjected to paired-end sequencing with a 300-cycles MiSeq Reagent kit (Illumina)
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and a MiSeq sequencer (Illumina). Removal of PhiX, low-quality (Q<30), and chimeric
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sequences, and assembly of the paired-end sequences were performed as described
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previously (Itoh et al., 2014). Briefly, the PhiX sequences that were present as
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contaminants in the sequence library were detected by homology search with the
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Greengenes database (DeSantis et al., 2006) and PhiX sequences using a
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Burrows-Wheeler Aligner, version 4.0.5 (Li and Durbin, 2009). Then the PhiX
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sequences were removed from the sequence library using self-written scripts. The
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paired-end sequences were joined together by a fastq-join tool in the ea-utils software
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package, version 1.1.2-301 (Aronesty, 2013). The connected sequences having scores of
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Q30 or greater were collected by command lines in the software package QIIME,
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version 1.7.0 (Caporaso et al., 2010). Using the program Mothur, version 1.31.2
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(Schloss et al., 2009), the Q30-filtered sequences were aligned, and then the chimeric
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structures were detected and removed from the library. The sequences in each sequence
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library were characterized phylogenetically using the QIIME software package
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(Caporaso et al., 2010). The abundance of the obtained sequences was determined in
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both the 13C mixture and the unlabeled mixture, and the statistical significance of their
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difference was calculated using the Student’s t-test.
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qRT-PCR of the heaviest fraction of RNA
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The 1H fractions of RNA with BDs of 1.791–1.806 g ml–1 were subjected to qRT-PCR
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with a SuperScript III Platinum SYBR Green one-step qRT-PCR kit (Life Technologies)
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and a real-time PCR detection system (MyIQ2; Bio-Rad). The concentrations of the 16S
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rRNAs from E. coli and total bacteria were determined. Briefly, the primers
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Gamma395f and Gamma871r were used for the specific detection of the class
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Gammaproteobacteria (Muhling et al., 2008), herein targeting the 16S rRNA from E.
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coli. In addition, the primer set 515f/806r was used to detect the 16S rRNA from total
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bacteria (i.e., E. coli and B. subtilis). In order to obtain a standard curve for
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quantification, the 16S rRNA gene from E. coli was amplified by PCR with the primers
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B27f and B907r, and its dilution series (10 to 108 copies μl–1) was prepared. The
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reaction mixture (total volume: 50 μl) of qRT-PCR contained the following: 25 μl of
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SYBR Green reaction mix, 1 μl of SuperScript III RT/Platinum Taq Mix, 0.2 μM of
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each primer, and 2 μl of template RNA. RT was carried out for 3 min at 56ºC (for E.
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coli) or at 52ºC (for total bacteria), and then qPCR was performed as an initial
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denaturation at 95ºC for 5 min, followed by 40 cycles of denaturation for 15 s at 95ºC,
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annealing for 30 s at 56ºC (for E. coli) or 52ºC (for total bacteria), and extension for 1
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min at 72ºC. Fluorescence was detected at the end of each extension step. A melting
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curve analysis was conducted between 72ºC and 95ºC to check the specific
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amplification of the 16S rRNA from E. coli during qRT-PCR with the primers
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Gamma395f and Gamma871r.
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