Library preparation and sequencing analysis were conducted at Bioengineering Lab. Co., Ltd. (Sagamihara, Japan). The concentration of the total RNA was measured using the Quantus Fluorometer with the QuantiFluor RNA system (Promega, Madison, WI, USA). The quality of the RNA was then analyzed using the 5200 Fragment Analyzer System (Agilent Technologies, Santa Clara, CA, USA). RNA sequencing libraries were prepared using the MGIEasy RNA Directional Library Prep Set (MGI Tech, Shenzhen, China) according to the manufacturer’s instructions. The concentration of the prepared library solution was determined using the Qubit 3.0 Fluorometer with the dsDNA HS Assay Kit (Thermo Fisher Scientific). The quality of the library was analyzed using the 5200 Fragment Analyzer System with the dsDNA 915 Reagent Kit (Agilent Technologies). Single-stranded circular DNA was prepared using the constructed library and the MGIEasy Circularization Kit (MGI Tech), and DNA nanoballs (DNBs) were prepared using the DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech). The 200 bp paired-end sequencing was performed using the DNBSEQ-G400 (MGI Tech). The short-read data were deposited in the Read Archive of DDBJ (accession number DRA014441).
Dnbseq g400rs high throughput sequencing kit
Manufactured by MGI Tech
Sourced in Japan
The DNBSEQ-G400RS High-throughput Sequencing Kit is a laboratory equipment product designed for high-throughput DNA sequencing. It provides the necessary reagents and consumables to perform large-scale DNA sequencing projects.
Automatically generated - may contain errors
Lab products found in correlation
Mgieasy rna directional library prep set, by MGI Tech (4 mentions)
Mgieasy circularization kit, by MGI Tech (4 mentions)
5200 fragment analyzer system, by Agilent Technologies (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Agilent hs rna kit, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Dsdna 915 reagent kit, by Agilent Technologies (2 mentions)
Quantifluor rna system, by Promega (1 mentions)
Quantus fluorometer, by Promega (1 mentions)
Dnbseq g400, by MGI Tech (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Dnbseq g400 instrument, by MGI Tech (1 mentions)
Random primer 6, by New England Biolabs (1 mentions)
Twist comprehensive viral research panel, by Twist Bioscience (1 mentions)
Nebnext ultra 2 non directional rna second strand synthesis module, by New England Biolabs (1 mentions)
Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (1 mentions)
Qubit dsdna hs assay kit, by Promega (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Ultrasonicator, by Covaris (1 mentions)
6 protocols using dnbseq g400rs high throughput sequencing kit
1
RNA-seq Library Preparation and Sequencing
2
RNA Directional Library Preparation and Sequencing
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The library was prepared using an MGIEasy RNA Directional Library Prep Set (MGI Tech Co, Ltd) following the manufacturer’s manual. To examine the prepared library quality, the circularized DNA was prepared using an MGIEasy circularization Kit (MGI Tech Co, Ltd) with the manufacturer’s guideline. After making DBA nanoball by a DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech Co, Ltd), the sequencing was performed using DNBSEQ-G400. The differential gene expression analysis was performed for transcripts aligned to more than ten reads using estimateSizeFactors function, estimateDispersions function, and nbinomWaldTest function in R package DESeq2.
3
Total RNA Extraction and RNA-Seq Analysis
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Total RNA was extracted from the samples using the RNeasy Mini Kit (Qiagen NV, Venlo, Netherlands). The integrity of the RNA was assessed using Agilent Technologies’ 2100 Bioanalyzer and RNA 6000 Nano Kit, which confirmed that all samples had an RNA integrity number of >9. The quality of the RNA was further assessed using the 5200 Fragment Analyzer System and the Agilent HS RNA Kit (Agilent Technologies). Subsequently, the DNA libraries were assembled using the MGIEasy RNA Directional Library Prep Set (MGI Tech). The quality of these libraries was assessed using the 5200 Fragment Analyzer System and the dsDNA 915 Reagent Kit (Agilent Technologies). The libraries were then circularized and converted into DNA nanoballs using the MGIEasy Circularization Kit and DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech). Finally, sequencing was performed on a DNBSEQ-G400 instrument (MGI Tech) with a read length of 2 × 100 bp.
4
Viral Metagenomics Using Twist Panel
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The extracted RNA was converted into double-stranded copy (c)DNA using the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA), NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs), and Random Primer 6 (random hexanucleotides; New England Biolabs). The obtained double-stranded (ds)DNA and DNA samples were subsequently converted to Illumina TruSeq-compatible libraries using Twist Library Preparation Enzymatic Fragmentation Kit (Twist Biosciences, CA, USA) and Twist Universal Adapter System-TruSeq Compatible (Twist Biosciences). We performed a single hybridization capture for each sample and enrichment using the Twist Comprehensive Viral Research Panel (Twist Biosciences). All libraries were converted into libraries for the DNBSEQ using the MGIEasy Universal Library Conversion Kit (App-A). Sequencing was performed using the DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech, Tokyo, Japan) in 100-base paired-end mode. Each read was subjected to KRAKEN2 analysis against the PlusPFP database containing archaea, bacteria, viral, plasmid, human, UniVec_core, protozoa, fungi, and plant33 (link),34 (link).
5
Whole Genome Sequencing of E. coli
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In total, 40 E. coli strains were subjected to whole genome sequencing (WGS). WGS was outsourced to Bioengineering Lab. Co., Ltd. (Kanagawa, Japan). First, the DNA was fragmented using an ultrasonicator (Covaris Inc, MA, USA) to a fragment length of 400 bp. The library preparation was conducted according to the manual using a MGIEasy Universal DNA Library PrepSet (MGI Tech Co., Ltd, Shenzhen, China). The concentration of the prepared library was measured using the Qubit 30 Fluorometer (Promega Co., WI, USA) and the Qubit dsDNA HS assay kit (Promega). The quality of the prepared library was confirmed using Fragment Analyzer and a dsDNA 915 Reagent Kit (Advanced Analytical Technologies, NY, USA). Circular DNA was prepared according to the manual using the created library and the MGIEasy Circularization Kit (MGI Tech Co.). A DNBSEQ G400RS high-throughput sequencing kit (MGI Tech) was used to prepare DNA nanoballs (DNBs) according to the manual. Sequencing analysis of the prepared DNBs was performed using DNBSEQ G400 under the condition of 2 × 150 bp.
6
RNA Sequencing Protocol for Transcriptome Analysis
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RNA sequencing was performed on our behalf by Bioengineering Lab (Kanagawa, Japan). Total RNA was extracted by RNeasy Mini Kit (Qiagen) with an additional step of on-column DNase I digestion. The RNA sample quality was checked with a 5200 Fragment Analyzer System (Agilent Technologies, Santa Clara, CA, USA) and Agilent HS RNA Kit (Agilent Technologies). Libraries for the sequencing were constructed using a MGIEasy RNA Directional Library Prep Set (MGI Tech, China), together with Dynabeads mRNA Purification Kit (Thermo Fisher Scientific) for removing the ribosomal RNA, according to the manufacturer’s instructions. After the measurement of concentrations with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and dsDNA HS Assay Kit (Thermo Fisher Scientific), the prepared library was circulated using a MGIEasy Circularization Kit (MGI Tech). DNA Nanoballs (DNBs) were subsequently prepared with a DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech). The sequence of these DNBs was analyzed on the DNBSEQ-G400 with 100-bp pair-end reads. The raw sequencing data were deposited in the DDBJ Sequence Read Archive (Kodama et al., 2012 (link)) (DRA accession number: DRA013757 ).
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