Applied biosystems 3130xl

One μL of restriction fragments was denatured at 95°C for 3 min in 10 μL Hi‐Di formamide (Cat. No. 4311320) containing 0.3 μL GeneScan 1200 LIZ size standard (Cat. No. 4379950; both from Applied Biosystems‐Thermo Fisher Scientific). Sizing of fluorescently‐labeled restriction fragments was performed on an automated capillary sequencer (Applied Biosystems 3130XL, Thermo Fisher Scientific). Peak patterns were analyzed with PeakScanner software (version 1.0, Thermo Fisher Scientific) and translated into a binary data matrix (presence‐absence).

KRAS mutations of the established cell line were examined following a previous report [25 (link)]. Briefly, according to the manufacturer’s instructions, reverse transcription of total RNA was performed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The KRAS was amplified with the forward primer KRAS_F (5′-TGT AAA ACG GCC AGT GTG TGA CAT GTT CTA ATA TAG TCA-3′) and the reverse primer KRAS_R (5′-CAG GAA ACA GCT ATG ACC GAA TGG TCC TGC ACC AGT AA-3′) using Platinum Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific, Fair Lawn, NJ, USA). To analyze the Sanger sequencing data, an identical primer set for the junction and the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Fair Lawn, NJ, USA) were used in the Applied Biosystems 3130xL by GENEWIZ (GENEWIZ, South Plainfield, NJ, USA).

The RNAs of wild-type and CA viruses were extracted from the supernatants using ISOGEN-LS (Nippon Gene, Tokyo, Japan). Reverse transcription of viral RNA was performed using primers containing the conserved sequences at the 3-prime ends of the viral segments using ReverTra Ace (TOYOBO, Osaka, Japan). Later, polymerase chain reaction (PCR) was conducted using specific primer pairs for each gene segment [32 (link)]. PCR products were purified using the Fast Gene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan) and sequenced directly using specific primers in an automated sequencer (Life Technologies Japan, Applied Biosystems 3130xl, Tokyo, Japan).

TALEN vectors were constructed as described by Takasu et al. (2014, 2016)^{48 (link),49 (link)}. The target site was selected within the coding region of the bmgstu2 gene, and the sequence around the target site was determined in the w1-pnd strain. TALEN was assembled using the Golden Gate TALEN and TAL Effector kit (Addgene, Cambridge, USA) and the TALEN backbone vector pBlue-TAL^{23 (link)}. The mRNA was in vitro synthesised using the mMESSAGE mMACHINE T7 kit (Ambion, Carlsbad, USA).
PITCh vector construction was carried out following the method described in Tsubota and Sezutsu (2017)⁵⁰ . Inverse PCR was carried out using the primers 5′-TGAGGCAAGCCTTGAAGTACCATTTGAACTAGCACCACCTGTTCCTGTAG-3′ and 5′-CGCGACAAGGCAGAGAGCGATGGAGGGCCACTCGAATTAGATCTTTGG-3′ against the pBachsp90GFP-3xP3DsRed plasmid^{24 (link),51 (link)}. The PCR product was self-ligated, and the inserted sequence was checked using Applied Biosystems 3130xl (Life Technologies) after cycle sequencing with BigDye Terminator V3.1 (Life Technologies, Carlsbad, USA).

Genomic DNA was isolated from adult moths by sodium dodecyl sulfate−phenol extraction (Ohshima and Suzuki 1977 (link)). The DNA was digested with NcoI and heated at 70° for 15 min to inactivate the enzymes. The digested DNA was purified using a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) or QIAquick Gel Extraction Kit (QIAGEN) and ligated using T4 DNA ligase (New England Biolabs, Ipswich, MA) at 4°overnight. The DNA was purified using a QIAquick PCR Purification Kit (QIAGEN) and was used as the template for PCR. PCR was carried out using the ExTaq polymerase (TaKaRa, Otsu, Japan) as follows: 95° for 2 min followed by five cycles of 95° for 30 sec, 65° for 30 sec and 72° for 2 min, 5 cycles of 95° for 30 sec, 60° for 30 sec and 72° for 2 min, 35 cycles of 95° for 30 sec, 55° for 30 sec and 72° for 2 min, and 72° for 10 min. The amplified fragments were sequenced using Applied Biosystems 3130xl (Life Technologies, Carlsbad, CA) after cycle sequencing with BigDye Terminator V3.1 (Life Technologies). The obtained sequence was subjected to a BLAST search in the silkworm genome database, KAIKObase (http://sgp.dna.affrc.go.jp/KAIKObase/) for the identification of the insertion site.

For R. toxicus FH-79, a shotgun DNA library was constructed for the 454 Junior (Roche) according to the manufacturer’s directions and three sequencing runs were performed. In addition, a library FH-79 was also constructed for the PacBio RSII (Pacific Biosciences); three SMRT cells were sequenced for FH-79 at the Washington State University Genomics Lab. The 454 sequence data was assembled using Lasergene Ngen v12.0 (DNAStar) and PacBio reads using Pacific Bioscience’s Hierarchical Genome-Assembly Process (HGAP) [14 (link)]; consensus sequences from the two methods were compared using Ngen. For R. toxicus FH-232, only a PacBio library was constructed and 3 SMRT cells were sequenced also at the Washington State University Genomics Lab; assembly was performed using Pacific Bioscience’s Hierarchical Genome-Assembly Process (HGAP) [14 (link)]. The putative tunicamycin gene cluster, vancomycin resistance genes, and 16S rDNA from FH-79 and the CRISPR region from FH-232 were resequenced by primer walking on an Applied Biosystems 3130XL (Thermo Fisher Scientific) to validate genome assembly.
The genome sequences presented here have been deposited in GenBank under the following accession numbers: R. toxicus FH-79 BioProject PRJNA312185 and BioSample SAMN04495682; R. toxicus FH-232 BioProject PRJNA312185 and BioSample SAMN06040670.

Cultures of R. toxicus FH-79 and FH-232 were obtained from Dr. Ian Riley (University of Adelaide, South Australia); additional information about their origins is presented in Table 1. R. toxicus was maintained on modified YGM (mYGM) [12 ]. One liter of this modified media contained yeast extract 2 g, glucose 1.25 g, K₂HPO₄ 0.25 g, KH₂PO₄ 0.25 g, MgSO₄·7H₂O 0.1 g, and agar 16 g. Cultures were incubated at 25°C unless otherwise noted; cryogenic stocks were stored in 15% glycerol at -80°C. DNA was extracted using a modified Marmur method [13 ] from 3 day old liquid cultures. DNA quality was estimated by OD_260/280 ratio as measured on a Nanodrop 2000 (Thermo Fisher Scientific) and only DNA with a ratio >1.6 was used for sequencing. Purity of the cultures used for DNA extraction was confirmed by plating 50 μl on mYGM and monitoring for growth of non-R. toxicus colonies. 16S rDNA was sequenced using an Applied Biosystems 3130XL (Thermo Fisher Scientific) to test purity of extracted DNA prior to genomic sequencing; only extracted DNA yielding a single 16S sequence was sequenced further.