Isolation of arenavirus was attempted with spleen sample. Vero cells were inoculated with the spleen homogenate and observed daily for cytopathic effect. For next-generation sequencing (NGS), library was constructed using Illumina Truseq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, United States) following the manufacturer’s instructions and sequencing was performed on an HiSeq 3000 sequencer (Illumina, San Diego, CA, United States). De novo assembly of NGS data was performed using Trinity version r2011-08-20 (Grabherr et al., 2011 (link)) and the resulting contigs were aligned to the non-redundant nucleotide database on NCBI. Contigs mapping significantly to the reference sequences were then retrieved and re-mapped to the full genome reference to generate a consensus sequence. The genome end sequence was amplified through the SMARTer® RACE 5′/3′ Kit (Takara). The recovered genome sequence was used for ORF prediction and aligned with those of representative members of mammarenavirus using ClustalW. Pairwise sequence similarities of the L/S segments and ORFs of the novel arenavirus to other mammarenaviruses were calculated using DNAstar. Phylogenetic inference of the aligned homologs of the four ORFs was done in MEGA version 7.0, using the ML approach and the Tamura–Nei model with a bootstrap of 1000 replicates.
Hiseq 3000 sequencer
Manufactured by Illumina
Sourced in United States, France, China
The HiSeq 3000 is a high-throughput DNA sequencing system designed for large-scale genomic research. It utilizes Illumina's sequencing-by-synthesis technology to generate high-quality sequencing data. The HiSeq 3000 is capable of producing up to 1.5 terabases of data per run, making it suitable for a wide range of applications, including whole-genome sequencing, exome sequencing, and transcriptome analysis.
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Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (7 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (6 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Hiseq 2000 sequencer, by Illumina (4 mentions)
High sensitivity dna chip, by Agilent Technologies (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Nextera xt index kit, by Illumina (3 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (2 mentions)
Smart seq v4 ultra low input rna kit for sequencing, by Takara Bio (2 mentions)
Nextera xt dna sample preparation kit, by Illumina (2 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Truseq rna sample prep kit, by Illumina (2 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Stranded rna seq library preparation kit, by Roche (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
80 protocols using hiseq 3000 sequencer
1
Isolation and Genome Characterization of Novel Arenavirus
2
Targeted Exome Sequencing Data Analysis
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Targeted enriched libraries were prepared using the SureSelectXT Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced with a paired-end protocol on a HiSeq 3000 Sequencer (Illumina, Little Chesterford, UK). The quality control of the raw sequence data, base quality scores, GC content and duplications were checked using java based FastQC software (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Sequence adaptors were removed with Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ ). Sequences were then aligned against the reference genome (hg19/GRCh37) using the Burrows–Wheeler Aligner BWA (v0.7.12-r1.39) [27 (link)]. SAM files were converted to BAM files with SAMtools then sorted by Picard tools (v2.5.0) (https://broadinstitute.github.io/picard/ ), which were also used to remove PCR duplicates. BAM files were realigned locally around the indels using the Genome Analysis Tool Kit GATK (https://gatk.broadinstitute.org/hc/en-us ) (v3.5) [28 (link), 29 (link)]. The GATK HaplotypeCaller function was used to call small indels and single nucleotide variants (SNVs) in genomic variant call format (g.VCF). The variant list was then annotated using Variant Effect Predictor (VEP) software [30 (link)].
3
Profiling Retinal Cell Transcriptomes
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Retinas (E17) were electroporated with either control or shSetd1a plasmid and cultured for 2 days. EGFP positive cells (∼3e4 cells for 1 sample) were collected by a cell sorter, FACS Aria II (BD Biosciences) as described.26 (link) Total RNA of three control and four shSetd1a samples was extracted using Sepasol RNA I Super G (Nacalai tesque) and quantified by using a 2100 Bioanalyzer (Agilent Technologies). Using 1 ng of total RNA, cDNA was prepared and amplified by PCR by SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara) according to the manufacturer's instructions. The RNA-Seq libraries were prepared using the amplified cDNA and Nextera XT DNA Sample Preparation Kit (Illumina). 36 bp of single read sequencing was conducted by HiSeq3000 sequencer (Illumina). The GEO accession number of RNA-Seq data is GSE154498.
4
Transcriptomic Analysis of Algal Culture
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A total of 50 ml of algal culture was sampled for transcriptomic analysis. Total RNA was extracted and sequenced as described by Carrier et al. (2014) (link). A library of RNA-seq was built for each sample. Poly(A) mRNA was isolated from total RNA using oligo (dT) magnetic beads [MicroPoly(A)Purist™ Kit; Ambion]. Two cDNA libraries were built according to the instructions of the manufacturer and sequenced with an Illumina HiSeq 3000 sequencer and with the GenoTool platform. We obtained an average of 2.6.17 and 3,2.17 reads per sample in the CL and DL experiments, respectively.
Raw reads of each sample were filtered using TrimGalore to remove known Illumina adapter sequences. Low-quality reads were excluded using a quality score threshold of 30 and a minimal length of 75 or 150 bases. The quality of reads was assessed using FastQC. Then, sequenced reads for each sample were aligned using Bowtie 2 in the QuasR package in R (Quantify and Annotate Short Reads in R). Each gene within the alignment was counted using the Rsubread package in R with the function feature Counts, based on T. lutea reference genome (Carrier et al., 2014 (link)). Gene counts were obtained for each sample and normalized with DESeq2.
Raw reads of each sample were filtered using TrimGalore to remove known Illumina adapter sequences. Low-quality reads were excluded using a quality score threshold of 30 and a minimal length of 75 or 150 bases. The quality of reads was assessed using FastQC. Then, sequenced reads for each sample were aligned using Bowtie 2 in the QuasR package in R (Quantify and Annotate Short Reads in R). Each gene within the alignment was counted using the Rsubread package in R with the function feature Counts, based on T. lutea reference genome (Carrier et al., 2014 (link)). Gene counts were obtained for each sample and normalized with DESeq2.
5
Grape DNA Isolation and Sequencing
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High quality genomic DNA was isolated from grape leaves using the method described in Chin et al. (2016) [53 (link)]. DNA purity was evaluated with a Nanodrop 2000 spectrophotometer (Thermo Scientific, Hanover Park, IL), quantity with a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA) and integrity by electrophoresis. For SMRT sequencing, SMRTbell libraries for the Zinfandel reference FPS clone 03 (Zin03) were prepared as described by Chin et al. (2016). For Illumina sequencing, DNA sequencing libraries for each of the fifteen Zinfandel clones were prepared using the Kapa LTP library prep kit (Kapa Biosystems) as described by Jones et al., (2014) [73 (link)]. Final libraries were evaluated for quantity and quality using a Bioanalyzer 2100 (Agilent Technologies, CA). Zin03 SMRTbell libraries were sequenced on a PacBio RS II and Illumina libraries were sequenced in 100 and 150 base-pair paired-end reads on an Illumina HiSeq3000 sequencer (DNA Technology Core Facility, University of California, Davis). Genome sequences of additional V. vinifera were used in this study, including long reads from Cabernet Sauvignon (NCBI BioProject PRJNA316730) and short reads from Cabernet Franc, Chardonnay, Merlot, Pinot Noir, and Sauvignon blanc (NCBI BioProject PRJNA527006).
6
Whole Exome Sequencing Protocol for CNV Analysis
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Three micrograms of genomic DNA were processed according to the Agilent SureSelect XT Library Prep protocol (Agilent Technologies, CA, USA). Sure Select Human All Exon V5 or V6 (Agilent Technologies) was used as the capture reagent. Sequencing was performed using a 150 bp paired-end protocol on an Illumina HiSeq 3000 sequencer (4981 and 4982) (Illumina, CA, USA). The resulting fastq files were aligned to the human reference genome (GRCh37) using BWA (44 (link)). The alignment was processed according to GATK best practice. Exome depth was used for CNV analysis according to the developers’ guidelines (22 (link)).
All genomic coordinates are based on the GRCh37 human reference genome. The reference gene sequence upon which SP6 mutation nomenclature is based is RefSeq transcript NM_199262.
The variant identified in this study has been submitted to the Leiden Open Variant Database athttp://dna2.leeds.ac.uk/LOVD/ variant ID: 0000000305.
All genomic coordinates are based on the GRCh37 human reference genome. The reference gene sequence upon which SP6 mutation nomenclature is based is RefSeq transcript NM_199262.
The variant identified in this study has been submitted to the Leiden Open Variant Database at
7
Comprehensive RNA Sequencing and qPCR Protocol
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Total RNA was extracted from harvested tissues using QIAGEN miRNeasy Mini Kit (217004, Qiazol) and RNase-Free DNase Set (79254).
cDNA libraries were prepared for the bulk RNA sequencing in triplicates according to the Smart-Seq2 protocol [53 (link)]. Subsequently, the libraries were pooled into one lane for sequencing at a HiSeq3000 sequencer (Illumina), using sequencing format of dual indexing and single 50 base-pair reads.
For qPCR, cDNA was synthesized using a cDNA Synthesis Kit (product no. 1708891, Biorad) and was analyzed by qPCR using SYBR Green (SsoAdvanced™ Universal SYBR Green Supermix, product no. 172-5274, Biorad). The expression levels of the genes were normalized to housekeeping genes β-Actin and Gapdh.
cDNA libraries were prepared for the bulk RNA sequencing in triplicates according to the Smart-Seq2 protocol [53 (link)]. Subsequently, the libraries were pooled into one lane for sequencing at a HiSeq3000 sequencer (Illumina), using sequencing format of dual indexing and single 50 base-pair reads.
For qPCR, cDNA was synthesized using a cDNA Synthesis Kit (product no. 1708891, Biorad) and was analyzed by qPCR using SYBR Green (SsoAdvanced™ Universal SYBR Green Supermix, product no. 172-5274, Biorad). The expression levels of the genes were normalized to housekeeping genes β-Actin and Gapdh.
8
Transcriptomic Analysis of Liver in PPARα Mice
9
Bisulfite and Oxidative Bisulfite Sequencing
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BS-seq and oxBS-seq were performed as previously described (Booth et al., 2013 (link)). 2 μg genomic DNA mixed with unmethylated lambda DNA was fragmented to an average size of 250 bp with S220 Focused-ultrasonicator (Covaris, USA). The randomly fragmented DNA was subsequently subjected to constructed libraries using TrueMethyl oxBS Module (Cat #0414, Tecan, USA). Briefly, the fragmented DNA was firstly treated with a combination of T4 DNA polymerase, Klenow fragment, and T4 polynucleotide kinase; then the blunted DNA fragments were added with sequencing spike-in control DNA duplex (containing SQ6hmC, SQ3hmC, SQ1hmC, SQC, SQmC, and SQfC) and were subsequently 3′ adenylated using Klenow fragment and ligated to 5mC adaptors using T4 DNA Ligase; the ligated products were treated either with 1 µL Oxidant Solution for oxBS-seq libraries or 1 μL Ultra-Pure Water for BS-seq libraries, followed by bisulfite conversion using Bisulfite Reagent. After PCR amplification using barcode-tagged primers, all 16 libraries (eight oxBS-seq libraries and eight corresponding BS-seq libraries) were pooled and sequenced on a HiSeq 3,000 sequencer (Illumina, USA) using 150 bp paired-end model.
10
Rat Podocyte miRNA Profiling
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One μg of total RNA isolated from the rat podocytes in primary culture was subjected to small RNA sequencing to comprehensively analyze miRNA expression. Small RNA libraries were constructed using the TruSeq Small RNA Library Preparation Kits (Illumina, San Diego, CA, USA) and analyzed on a HiSeq-3000 sequencer (Illumina) at the Genome Information Research Centre of Osaka University. Raw reads obtained from the sequencing analysis were trimmed using Cutadapt v1.9.2 and subjected to miRNA-derived read counting and annotation using CLC Genomics Workbench 11.0.1 (Qiagen, Venlo, The Netherlands).
The reads obtained in this analysis and related metadata were deposited in the DNA Data Bank of Japan Sequence Read Archive (DRA) under the accession number DRA013173.
The reads obtained in this analysis and related metadata were deposited in the DNA Data Bank of Japan Sequence Read Archive (DRA) under the accession number DRA013173.
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