Genequant 100

Total RNA was extracted from each sample excised with LMD using a micro RNA Extraction kit (Qiagen, Valencia, CA). After the isolation steps, we quantified RNA using a GeneQuant100 (GE Healthcare Ltd., Amersham, UK) and equalized each sample. The cDNA was synthesized from total RNA with SuperScript^® VILOTM Master Mix (Life technology, Carlsbad, CA), following manufacturers instruction. For quantitative RT-PCR, primers for Slc26a4 or GAPDH (Takara Bio., Otsu, Japan) and templates were mixed with SYBR^® Green PCR Master Mix (Life technology). The cDNA was amplified for 40 cycles of denaturation for 15 sec at 95 °C and annealing for 1 min at 60 °C, using a Takara thermal cycler Dice system (model TP960). The relative gene expression of Slc26a4 and of GAPDH was calculated by the standard curve method. Transcript levels of Slc26a4 were normalized to GAPDH.

Total RNA was extracted from the kidney, lung, and heart with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA concentration was determined by the absorbance read at 260 nm (GeneQuant 100, GE Healthcare UK Ltd, Chalfont St Giles, Buckinghamshire, UK). The primers and TaqMan probes for tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and glutaraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were purchased from a commercial laboratory (Applied Biosystems, Foster City, CA, USA). The mRNA expressions of TNF-α, IL-1β, and IL-6 were determined by real-time polymerase chain reaction (PCR). cDNA was synthesized using TaqMan reverse transcription reagents (Applied Biosystems, Roche Molecular Systems, Inc., Branchburg, NJ, USA) and quantified using a thermal cycler (PC707, ASTEC Co., Ltd., Minato-ku, Japan). TaqMan real-time PCR was performed using an ABI 7900HT (Applied Biosystems, Foster City, CA, USA). TaqMan rat GAPDH was used as an internal control and relative gene expression values were determined using the 2^−ΔΔCT method [21 ].

The phage fractions derived from A. pasteurianus NBRC 109446, A. orleanensis NBRC 3170, Acetobacter sp. ATCC 21760, K. xylinus NBRC 13772, K. xylinus NBRC 13773, and K. maltaceti NBRC 14815 were prepared as described above. The fractions were treated with 20  $\mathrm{\mu }$ g/mL DNase I (Sigma-Aldrich Corp, St. Louis, MO, USA) and 10  $\mathrm{\mu }$ g/mL RNase (Sigma-Aldrich) at 37 °C for 30 min. Their phage genomic DNAs were purified with Phage DNA isolation kit (Norgen Biotek Corp., Ontario, Canada) according to manufacturer’s instruction. Quality and quantity of the purified phage genomic DNAs were verified with GeneQuant 100 (GE Healthcare UK Ltd, Buckinghamshire, England), and agarose gel electrophoresis analyses. Genome sequence analysis with next generation sequencing was performed using Illumina MiSeq with 150-bp paired-end reads by Hokkaido System Science Co. Ltd. (Sapporo, Japan). DFAST^{26 (link)}, RAST^{27 (link)}, 2ndFind (http://biosyn.nih.go.jp/2ndfind/), PHAST^{15 (link)} was used for the annotation of phage genomes. Genetyx ver. 13 (GENETYX Corporation, Tokyo, JAPAN) was used for local BLAST searches using draft genome sequences. Schematic representation of gene organization was drawn with drawGeneArrows3 (http://www.ige.tohoku.ac.jp/joho/index.html). The prediction of the tRNA gene was performed with tRNAscan-SE²⁸. Multiple alignment was carried out by CLUSTAL W²⁹.

Total genomic DNA was extracted using the conventional phenol-chloroform method as described previously with some modification (15 ). Briefly, a pool of approximately 30 muscle larvae of Trichinella was placed in a 1.5 ml microcentrifuge tube and mixed with 500 μl of lysis buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 20 mM EDTA pH 8.0 and 1% SDS). Following sonication using a T10 UltraTurrax tissue disperser (IKA-WERK, Staufen, Germany), 10-μl proteinase K (20 mg/ml; Qiagen) was added and the mixture was incubated at 55 °C for overnight. proteinase K was inactivated at 100 °C for 30 min. Purification of DNA was done by phenol and phenol-chloroform extraction, followed by ethanol precipitation. DNA concentrations were determined by spectrophotometrically by GeneQuant 100 (GE Healthcare Life Science). DNA was resuspended with 30 μl distilled water and frozen at −20 °C for use in PCR amplifications.

The RNA isolation was performed from blood leukocytes collected in a vacuum tube containing the EDTA anticoagulant. A sample volume of 20 μL was used for RNA extraction, according to the protocol using the GeneJET RNA Purification Kit (Thermo Scientific®, USA), in line with the manufacturer's recommendations. The total RNA was resuspended in 20 μL of RNase-free water and quantified using a GeneQuant 100 spectrophotometer (GE Healthcare, Wisconsin, USA); after that, cDNA was synthesized from 200 μg of total RNA, using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific®, USA), according to the manufacturer's recommendations. In short, approximately 200 μg of total RNA was reverse transcribed in a final volume of 20 μL of reaction. The resulting cDNA was stored at −30°C until real-time PCR analysis.

To obtain a complete cDNA libary, the mycelia of C. gloeosporioides ES026 cultured for 5, 7 and 12 days were collected and immediately mixed and stored in liquid nitrogen until further processing. Total RNA was extracted from 1 g of mycelia using the Total RNA Kit I (Omega Bio-Tek, Norcross, GA, USA) and treated with DNase I for 30 min at 37°C to remove residual DNA. The RNA concentration was measured with a GeneQuant 100 spectrophotometer (GE Healthcare, Chalfont St. Giles, UK). The RNA sample was sent to Bio-Briod (Wuhan Genomic Institute, China) for RNA sequencing. The sequencing was performed with an Illumina HiSeq 2000 (HiSeq 2000 TruSeq SBS Kit v3-HS).