Hiseq 2500 sequencing system

Manufactured by Illumina

Sourced in United States, China, Germany

The HiSeq 2500 is a high-throughput DNA sequencing system from Illumina. It is capable of generating up to 1 terabase of sequencing data per run, making it suitable for a wide range of applications, including genome sequencing, transcriptome analysis, and targeted resequencing. The system utilizes Illumina's proprietary sequencing-by-synthesis technology to deliver accurate and reliable results.

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Lab products found in correlation

Agilent 2100 bioanalyzer, by Agilent Technologies (17 mentions) Trizol reagent, by Thermo Fisher Scientific (12 mentions) 2100 bioanalyzer, by Agilent Technologies (11 mentions) Trizol, by Thermo Fisher Scientific (11 mentions) Rneasy mini kit, by Qiagen (9 mentions) Truseq rna sample preparation kit, by Illumina (6 mentions) Agilent bioanalyzer 2100 system, by Agilent Technologies (6 mentions) Bioanalyzer, by Agilent Technologies (6 mentions) Truseq rna library prep kit, by Illumina (5 mentions) Agilent bioanalyzer, by Agilent Technologies (5 mentions) Truseq rna sample preparation kit v2, by Illumina (5 mentions) Truseq stranded total rna library prep kit, by Illumina (5 mentions) Truseq rna library prep kit v2, by Illumina (5 mentions) Mirvana mirna isolation kit, by Thermo Fisher Scientific (4 mentions) Nextera dna sample preparation kit, by Illumina (4 mentions) Truseq rna library preparation kit, by Illumina (4 mentions) Ovation rna seq system v2, by Tecan (4 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions) Truseq stranded mrna sample prep kit, by Illumina (3 mentions) Qiaamp dna mini kit, by Qiagen (3 mentions)

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HiSeq 2500 (12028 citations) HiSeq 2500 system (1300 citations) HiSeq system (379 citations) HiSeq sequencing (173 citations) HiSeq 2500 sequencing (93 citations) HiSeq sequencing system (72 citations) HiSeq 2500 Ultra-High-Throughput Sequencing System (15 citations) HiSeq 2500 Sequencing Systems Platform (3 citations) HiSeq 2500 DNA sequencing system (2 citations) HiSeq 2500 and 3000 sequencing system (2 citations)

251 protocols using hiseq 2500 sequencing system

Genome Sequencing of Arabidopsis Thaliana Demes

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Leaf material from 74 individuals from 19 A. thaliana demes (four individuals from each deme except for two individuals from JBB) (Dataset S1) were used for DNA extraction (SI Appendix, section S4) and genome resequencing. Genome resequencing was performed on an Illumina HiSeq 2500 sequencing system in paired-end mode with data processing described in SI Appendix, section S5.
Frozen leaf material from one individual of the JBB, T11, and PA10 demes was used to perform de novo genome assemblies with linked reads generated with the 10× Genomics Chromium platform and Supernova assembler (SI Appendix, section S6). Samples S1 (T11), S2 (JBB), and S12 (PA10) were sequenced on an Illumina HiSeq 2500 sequencing system in rapid run mode (250-bp or 150-bp paired-end sequences). Fragment sizes were assessed using a Q-card (OpGen Argus optical mapping system) and the Genomic DNA TapeStation assay (SI Appendix, Table S5); assembly metrics are described in SI Appendix, Table S6.

Busoms S., Paajanen P., Marburger S., Bray S., Huang X.Y., Poschenrieder C., Yant L, & Salt D.E. (2018). Fluctuating selection on migrant adaptive sodium transporter alleles in coastal Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America, 115(52), E12443-E12452.

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Transcriptome Analysis of Heart Failure

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Sequencing was performed for all patients on the Ion Torrent PGM utilizing the Ion 316 chip. Coverage analysis as well as mapping the reads and alignment was done using the Ion Torrent Browser Suite™. Gene expression profiles from the PGM sequencer were validated with a subsequent population by performing RNAseq with the Illumina Hiseq2500 sequencing system. RNAs from 7 non failing and 3 failing samples were sequenced using the Illumina Hiseq2500 sequencing system (University of Nebraska Medical Center, DNA core facility). All paired end sequencing adaptors were trimmed using fqtrim and any reads shorter than 36 bases were discarded. A quality filter using fqtrim −q 5 was then applied to the reads. Paired end reads that passing those filters were mapped to Homo sapiens (release 37) reference sequence (GRCh37/hg19); using BowTie2 and TopHat2. The raw read counts per gene were generated using the Rsubread package.

Dhar K., Moulton A.M., Rome E., Qiu F., Kittrell J., Raichlin E., Zolty R., Um J.Y., Moulton M.J., Basma H., Anderson D.R., Eudy J.D, & Lowes B.D. (2016). Targeted myocardial gene expression in failing hearts by RNA sequencing. Journal of Translational Medicine, 14, 327.

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RNA-Seq of MCF-7 Septin 9 Overexpression

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A total of 12 samples were used for RNA-Seq analysis (three biological replicates from each of the MCF-7 isoforms and control cells). Sequencing libraries for the Illumina 2500 platform were created from the polyadenylated fraction of RNA from each cell line. mRNA was then isolated with Dyna1 oligo-dT beads (Invitrogen, Carlsbad CA) from 10μg of total RNA. The mRNA was randomly fragmented using the RNA fragmentation kit from Ambion. First-strand cDNA synthesis was performed using random primers and SuperScriptII reverse-transcriptase (Invitrogen, Carlsbad CA). This was followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H (Invitrogen, Carlsbad CA). The Illumina adaptor was ligated to the ends of the double-stranded cDNA fragments and a 200bp size-selection of the final product was performed by gel-excision, following the Illumina-recommended protocol. Template molecules of 200bp cDNA with the adaptor attached were enriched by PCR to create the final library. Libraries were pooled and sequenced in two lanes on the Illumina HiSeq (2500) Sequencing System (Illumina, San Diego, CA) using 150bp reads at the New York Genome Center (New York, NY). Sequences in fastq format as well as normalized data files have been deposited into Gene Expression Omnibus (GEO)^{100 (link)} (Project ID# GSE119449 entitled: ”Septin 9 over expression in MCF7”).

Marcus J., Bejerano-Sagie M., Patterson N., Bagchi S., Verkhusha V.V., Connolly D., Goldberg G.L., Golden A., Sharma V.P., Condeelis J, & Montagna C. (2019). Septin 9 isoforms promote tumorigenesis in mammary epithelial cells by increasing migration and ECM degradation through metalloproteinase secretion at focal adhesions.. Oncogene, 38(30), 5839-5859.

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RNA-Seq of MCF-7 Septin 9 Overexpression

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mRNA and Small RNA Sequencing of Transfected Cells

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For mRNA sequencing, total RNA was extracted from the transfected MDA-MB-231 cells with TRizol reagent (Invitrogen) following the manufacturer instructions. Approximately 5 μg total RNA of each sample was used for the cDNA reverse transcription. After quantitative analysis and quality inspection, Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) was used to establish sequencing libraries. For small RNA sequencing, total RNA was extracted using the mirVana TM miRNA Isolation Kit (Ambion, Austin, TX) following the manufacturer’s instructions. For small RNA library establishment and deep sequencing, the 135–170 nt size range of RNA was enriched by polyacrylamide gel electrophoresis. RNA from each sample was subject to DNA sequencing with an Illumina HiSeq 2500 Sequencing System according to the manufacturer’s instructions.

Zhou J., Wan F., Wang Y., Long J, & Zhu X. (2019). Small RNA sequencing reveals a novel tsRNA-26576 mediating tumorigenesis of breast cancer. Cancer Management and Research, 11, 3945-3956.

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RNA Sequencing of WNT10B-Treated Dermal Papilla Cells

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Total RNA from WNT10B-treated and control DPCs was extracted using TRIzol™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. The total RNA quantity and purity were analyzed using Bioanalyzer 2100 (Agilent Technologies, Inc.) and RNA 6000 Nano LabChip Kit (Agilent Technologies, Inc.). RNA samples with an RNA integrity number > 7.0 were used for subsequent experimentation. The Illumina TruSeq RNA Sample Preparation kit (Illumina, Inc.) was used to construct sequencing libraries. Sequencing was performed using an Illumina HiSeq 2500 Sequencing system (Illumina, Inc.) by Shanghai YingBiotech Co., Ltd. Each group was analyzed in triplicate.

Zhou Q., Song Y., Zheng Q., Han R, & Cheng H. (2019). Expression profile analysis of dermal papilla cells mRNA in response to WNT10B treatment. Experimental and Therapeutic Medicine, 19(2), 1017-1023.

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Gut Microbiome Profiling via 16S rRNA Sequencing

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The feces of each animal were collected in a sterile EP tube and stored at −80°C for DNA extraction. Microbial genomic DNA was extracted from stool samples using the E.Z.N.A^® Soil DNA Mini Kit (R6825, Omega Bio-Tek, Norcross, GA, United States), in accordance with the manufacturer’s protocols. The concentrations and purity of the resultant DNA samples were assessed using a NanoDrop™ system (NanoDrop, United States), following which the DNA was stored at −80°C for further tests.
The 16S rDNA gene was amplified using PCR with primers 16S-F (5′-CCTACGGGNGGCWGCAG-3′) and 16S-R (5′-GGACTACHVGGGTATCTAAT-3′), which target the V3–V4 region of the bacterial 16S rDNA gene. PCR reactions were performed in triplicate with Phusion^® High-Fidelity PCR Master Mix (New England Biolabs) using template DNA. PCR products were purified using the AxyPrep™ DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States), according to the manufacturer’s instructions and quantified using ABI StepOnePlus™ Real-Time PCR System (Life Technologies, Foster City, CA, United States). The purified PCR products were pooled in an equimolar ratio and paired-end sequenced (2 × 250) on an Illumina HiSeq 2,500 Sequencing System (Illumina, San Diego, CA, United States) according to standard protocols (Caporaso et al., 2012 (link)).

Li J., Cao Y., Lu R., Li H., Pang Y., Fu H., Fang G., Chen Q., Liu B., Wu J., Zhou Y, & Zhou J. (2021). Integrated Fecal Microbiome and Serum Metabolomics Analysis Reveals Abnormal Changes in Rats with Immunoglobulin A Nephropathy and the Intervention Effect of Zhen Wu Tang. Frontiers in Pharmacology, 11, 606689.

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Transcriptomic Analysis of hPSCs at Varying Oxygen Levels

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RNA from MEL1 and MEL2 hPSC cultured at 5% and 20% oxygen was isolated using the RNeasy Plus Mini Kit (Qiagen). The RNA integrity numbers (RIN) of 10 for all samples were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was analyzed on the Illumina HiSeq 2500 Sequencing System (Illumina) at the Australian Genome Research Facility (AGRF) using 1 μg of total RNA as input. Sequencing was performed with 12 samples per lane, using 50 bp single-end reads. The quality of resultant raw FASTQ data files was assessed with FastQC. Data files were uploaded to the Galaxy platform [59 (link)] at https://usegalaxy.org/ and processed using the following pipeline: FASTQ Groomer to convert raw Illumina FASTQ files to Sanger Quality score format, Trim Galore! to trim the 13 bp of Illumina standard adapters, RNA STAR to align reads to the UCSC GRCh37/hg19 human reference genome, featureCounts to measure gene expression, and DESeq2 to identify DEGs. GO analysis of Biological Function terms and KEGG pathways were determined using DAVID. DEGs were submitted to NetworkAnalyst [38 (link)] to visualize zero-order protein-protein interaction (PPI) networks using the STRING interactome with a confidence score cut-off of 900.

Lees J.G., Cliff T.S., Gammilonghi A., Ryall J.G., Dalton S., Gardner D.K, & Harvey A.J. (2019). Oxygen Regulates Human Pluripotent Stem Cell Metabolic Flux. Stem Cells International, 2019, 8195614.

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Dental Epithelium RNA Extraction and Sequencing

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Animals (∼1-y-old adult MZ and LF males) were killed and immediately dissected for RNA extraction. A ribbon ∼1 mm × 10 mm of epithelium was removed labial to the outer row of teeth from the dentary of experimental animals using a no. 12 scalpel blade. The extraosseous soft tissue was removed from the entire jaw to reduce the risk of TBs containing epithelium carryover. The bone was then shaved down using a scalpel to expose the bony crypts, and intraosseous RT were extracted with fine forceps. Extracted tissue was quickly placed in RNAlater RNA Stabilization Reagent (Qiagen). Tissues were frozen in liquid nitrogen, homogenized using a mortar and pestle, and placed in TRIzol. Following standard chloroform extraction, RNeasy mini columns (Qiagen) were utilized to purify RNA for storage at −80 °C. Total RNA was quantified using Qubit (Molecular Probes) and quality analyzed using the Agilent 2100 Bioanalyzer System for RNA library preparation. RNA input was normalized to 1 µg, and libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina-Kit A). Libraries were again quantified, quality assessed, and normalized for sequencing on the HiSeq 2500 Illumina Sequencing System.

Bloomquist R.F., Fowler T.E., An Z., Yu T.Y., Abdilleh K., Fraser G.J., Sharpe P.T, & Streelman J.T. (2019). Developmental plasticity of epithelial stem cells in tooth and taste bud renewal. Proceedings of the National Academy of Sciences of the United States of America, 116(36), 17858-17866.

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Clenbuterol-Induced Skeletal Muscle Transcriptome

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For chronic clenbuterol studies, lean WT mice (12-week-old males) consumed clenbuterol (30 mg/l) or regular drinking water for 5 days. Subsequently, quadriceps muscles were isolated from overnight (15 h) fasted or freely fed mice. Control and SKM-Rictor-KO mice were treated in the same fashion, but gastrocnemius muscle samples were collected from fed mice only. Total RNA was prepared from frozen tissues as described above. The RNA quality of all samples was analyzed on the Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip. cDNA libraries were generated using New England Biolabs Ultra II kit following the manufacturer’s instructions. Indexed libraries of good quality (RNA Integrity Number > 8) were pooled and used for sequencing on a HiSeq 2500 Illumina Sequencing System. Single end 50 bp reads were mapped to mouse genome mm10. Analysis of differentially expressed genes was performed on the Genomatix Genome Analyzer using the DESeq2 (two-tailed Wald’s test) software package. Functional enrichment and pathway analysis of differentially expressed genes was performed using Qiagen IPA software and ToppGene Suite^{64 (link)}.

Meister J., Bone D.B., Knudsen J.R., Barella L.F., Velenosi T.J., Akhmedov D., Lee R.J., Cohen A.H., Gavrilova O., Cui Y., Karsenty G., Chen M., Weinstein L.S., Kleinert M., Berdeaux R., Jensen T.E., Richter E.A, & Wess J. (2022). Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells. Nature Communications, 13, 22.

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