The cDNA library was constructed with NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7530) and NEBNext Multiplex Oligos for Illumina (NEB, E7500) according to the manufacturer’s instructions. Briefly, approximately 250∼300 bp RNA inserts were fragmented from the enriched mRNA. Then, the first-strand cDNA and second cDNA were synthesized from them. End-repair/dA-tail and adaptor ligation were performed for the double-stranded cDNA. Agencourt AMPure XP beads (Beckman Coulter, Inc.) were used to isolate the suitable fragments, and enriched by PCR amplification. Finally, sequencing of the constructed cDNA libraries were performed on an Illumina HiSeqX sequencing platform.
Hiseq x sequencing platform
Manufactured by Illumina
Sourced in United States
The HiSeq X sequencing platform is a high-throughput DNA sequencing system designed for large-scale genome sequencing projects. It utilizes sequencing-by-synthesis technology to generate massive amounts of sequence data with high accuracy and speed.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy micro kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Truseq dna pcr free library preparation kit, by Illumina (1 mentions)
Truseq dna sample preparation guide, by Illumina (1 mentions)
Gentra puregene kit, by Qiagen (1 mentions)
Mrna seq v3 library prep kit, by Vazyme (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Gexscope single cell rna library kit, by Singleron Biotechnologies (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Rna screen tapes, by Agilent Technologies (1 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions)
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
Truseq dna pcr free library prep kit, by Illumina (1 mentions)
Truseq dna single indexes, by Illumina (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Truseq dna pcr free kit, by Illumina (1 mentions)
Blood and tissue kit, by Qiagen (1 mentions)
Enzymatic methyl seq kit, by New England Biolabs (1 mentions)
Gexscope single cell rna library kit tissue, by Singleron Biotechnologies (1 mentions)
22 protocols using hiseq x sequencing platform
1
Illumina cDNA Library Construction
2
Genetic Diversity of Ryukyu Islanders
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A total of 50 Ryukyu islanders (Okinawa, n = 25; Miyako, n = 25) participated in this study. Okinawans came from Okinawa Island. The Miyako islanders were identified from the Okinawa Bioinformation Bank and came from the four Miyako islands (Miyako-jima: n = 17; Ikema: n = 2; Irabu: n = 4; Tarama: n = 2). The geographic locations of each population sampled in this study are shown in Fig. 1 . All subjects provided written informed consent to participate in this research. We confirmed by interview that all four grandparents of each participant were living on the respective islands. DNA samples were collected from blood or saliva of each participant. This study was approved by the ethical committees at the University of the Ryukyus and the University of Tokyo.
DNA was extracted from blood or saliva by SRL, Inc. (Tokyo, Japan). WGS was conducted at Macrogen Japan Corp. (Tokyo, Japan). DNA quality was assessed using the picogreen method, and the condition of the DNA was assessed by gel electrophoresis. A WGS library was constructed using a TruSeq DNA PCR-Free Library Preparation kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocols. We sequenced the DNA using 2 × 150-bp paired-end reads on a HiSeq X sequencing platform (Illumina Inc.).
DNA was extracted from blood or saliva by SRL, Inc. (Tokyo, Japan). WGS was conducted at Macrogen Japan Corp. (Tokyo, Japan). DNA quality was assessed using the picogreen method, and the condition of the DNA was assessed by gel electrophoresis. A WGS library was constructed using a TruSeq DNA PCR-Free Library Preparation kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocols. We sequenced the DNA using 2 × 150-bp paired-end reads on a HiSeq X sequencing platform (Illumina Inc.).
3
Whole Genome Sequencing of Translocation Junctions
4
Whole Genome Sequencing of Dogs
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The four GS samples selected for whole genome sequencing (WGS) (2 PRA cases and both unaffected parents; study accession number PRJEB32096) were normalized to 25 nanograms per μL (ng/μL), and 1000 ng was sent for sequencing, outsourced to Edinburgh Genomics, UK. Illumina sequencing of a TruSeq Nano library on a HiSeq X sequencing platform generated a dataset of approximately 30× coverage of the dog genome. Reads were aligned to the canine reference genome (CanFam3.1) using BWA-mem, variant calls were made using GATK (HaplotypeCaller) and base quality score recalibration, and indel realignment and duplicate removal were performed [37 (link)]. Single nucleotide polymorphism (SNP)and small insertion/deletion (INDEL) discovery was performed using standard hard filtering parameters or variant quality score recalibration according to GATK Best Practices recommendations [38 (link),39 ]. Sequencing reads and variants were visualized in IGV. Genomic Variant Call Format (VCF) files from 116 canine WGS were combined by HaplotypeCaller into a multi-sample VCF file. Variant Effect Predictor (VEP) was run on the merged VCF file for cross-genome analysis.
5
RNA-seq Analysis of Young Spikelet Hulls
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At the heading stage, TQ and SMS with a young spikelet hull length of 5 cm-10 cm were sampled and collected 3 times, quickly placed in tin foil, frozen in liquid nitrogen, and then stored in a refrigerator at −80 °C until further use. For RNA-seq, the total RNA of the fresh young spikelet hulls was extracted using Trizol, according to the manufacturer’s protocol. Three biological replicates were used for each sample. A total of 2.0 μg of RNA per sample was used to construct cDNA libraries using an mRNA-seq V3 Library Prep Kit (Vazyme, Nanjing, China), according to the manufacturer’s instructions. A Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) was used to quantify and assess the quality of the RNA samples and cDNA libraries. Paired-end 2 × 150-base sequencing was performed on an Illumina HiSeq X sequencing platform.
6
Identifying Soybean Seed Protein QTLs
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Firstly, the QTLs detected for more than two years were selected. Statistically significant QTLs associated with soybean seed protein content were identified by examining the genotypes within the QTL regions using SNP markers. We performed the genome sequencing of SD, YS2035, and IM using the Illumina Hiseq X sequencing platform (Illumina, San Diego, CA, USA). Reads were mapped using Bowtie 2 (v2.2.4) and variants were called with Freebayes (v1.3.4). After verifying tri-parent SNP selection based on the soybean reference genome, only genes that exhibited differences from SD were screened for SNPs in YS2035 and IM. The QTL regions were further investigated using SoyBase (www.soybase.org (accessed on 5 September 2023)) to identify the candidate genes. Annotated information on the candidate genes was obtained from the soybean reference genome (Wm82. a4. v1). The candidate genes were presented based on their gene descriptions and SNP variations within the QTL regions.
7
Whole-genome sequencing of Quechua Andeans
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We performed high-coverage whole-genome sequencing (WGS) analysis of DNA from 40 unrelated Quechua Andean men and women selected at random from the 224 recruited Andeans in this study on the Illumina HiSeqX sequencing platform (average and minimum call rate, 92.4 and 91.7%, respectively). An average of 2.86 Gb of sequencing data was aligned for each genome. We obtained an average of 30× coverage (76.0% of the genome) with 99.5% exonic coverage. Approximately 3.76 million of the total 12.51 million single-nucleotide variants (SNVs) were identified across each genome with 14.1% previously unidentified SNVs. For each sample, we identified an average of 188,852 SNVs across the whole exome, of which 0.29% were novel. As determined by Annotate Variation (ANNOVAR), there were 41,236 synonymous and 48,057 nonsynonymous SNVs (10,600 missense, 82 stopgain, and 10 stoploss on average).
8
Single-cell RNA-seq Library Preparation
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The cell concentration of the single-cell suspension was maintained at 1 × 105 cells/ml and loaded onto a microfluidic chip. Single-cell RNA-seq (scRNA-seq) libraries were constructed using the GEXSCOPETM Single Cell RNA Library Kit (Singleron Biotechnologies) (12 (link)). Individual libraries were diluted to 4 nM and sequencing was completed on the Illumina HiSeq X sequencing platform, selecting the 150-bp double-end mode.
9
Single-cell RNA-seq of minced tissue
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Fifty to one hundred milligrams of tissue was minced and dissociated with 2 mL of GEXSCOPE™ tissue dissociation solution (Singleron, Nanjing, China) at 37 °C for 15 min. After dissociation into a single cell suspension, the number of cells was counted using trypan blue staining. The cell concentration was adjusted to 1 × 105 cells/mL; then cells were added to the microfluidic chip and sequenced using the Illumina HiSeq X sequencing platform. The sequencing mode was 150 bp paired-end sequencing. Cell type identification and cluster analysis were performed on RNA-seq data using the Seurat program (http://satijalab.org/seurat/ , R package, v.3.0.1).
10
Transcriptome profiling of IgA nephropathy
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Total cellular RNA was extracted from CD19 positive B lymphocytes using the using a miRNeasy Micro Kit (Qiagen) following the manufacturer's instructions. The RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Electron Corporation) at 260/280 nm. The NGS libraries were prepared using VAHTS mRNA‐seq v2 Library Prep Kit for Illumina (Vazyme). RNA samples in each group were sent for mRNA deep sequencing on an Illumina HiSeq X sequencing platform with 6GB reads (Illumina). Differential expression gene analysis was performed using R v3.2.2. The log2 transformation was used to obtain the standardized expression values. The t test was used to detect differentially expressed genes between patients with IgAN and controls. Significantly up‐regulated genes were defined by as a logarithmic transformed fold‐change (FC) > 0.26 and P value ≤.05. Significantly down‐regulated genes were defined by a logFC ≤ 0.26 and P value ≤.05. The data discussed in this publication have been deposited in NCBI sequence read archive (SRA) and are accessible through SRA Series accession number PRJNA563895.
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