Highseq 2000

Manufactured by Illumina

Sourced in United States

The HiSeq 2000 is a high-throughput DNA sequencing system designed for large-scale genomic projects. It uses reversible terminator-based sequencing technology to generate up to 600 gigabases of DNA sequence data per run. The system is capable of sequencing up to eight samples in parallel, providing cost-effective and efficient data generation for a variety of applications, including whole-genome sequencing, exome sequencing, and transcriptome analysis.

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HighSeq (15 citations)

38 protocols using highseq 2000

Transcriptomics of Bamboo Flowering Stages

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Tag library preparation for samples of the four different flowering developmental periods (F1, F2, F3 and F4) was performed in parallel using the Illumina gene expression sample preparation kit. Transcriptome data of nonflowering moso bamboo leaves (CK) were regarded as the reference gene database, and the four libraries were sequenced using the Illumina high‐seq 2000 at Beijing Genomics Institute (BGI) (Shenzhen, Guangdong Province, China) (Gao et al., 2014).
Total RNA was extracted from the frozen samples (CK, F1, F2, F3 and F4) using the Trizol reagent (Invitrogen, Carlsbad, California, USA), according to the manufacturer's instructions. Five small RNA libraries were constructed for moso bamboo, and the Illumina high‐seq 2000 sequencing was carried out by the Beijing Genomics Institute (BGI) (Shenzhen, Guangdong Province, China) (Gao et al., 2015).
KEGG pathway analysis was performed after we obtained the sequencing data. Key regulation pathways and genes were selected according to expression abundance. Hierarchical clustering of expressional data was carried out using the Cluster 3.0 and Treeview programs (Eisen et al., 1998).

Ge W., Zhang Y., Cheng Z., Hou D., Li X, & Gao J. (2016). Main regulatory pathways, key genes and microRNAs involved in flower formation and development of moso bamboo (Phyllostachys edulis). Plant Biotechnology Journal, 15(1), 82-96.

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rRNA Depletion and RNA-seq Library Preparation

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rRNA was depleted with Ribo-Zero Gold rRNA Removal kit (Illumina Inc.) and sequencing libraries were prepared with Nextera (Illumina; ventral hippocampus) or ScriptSeq v2 (Epicentre; medial prefrontal cortex) RNA-seq library preparation kits. Sequencing was conducted on HighSeq 2000 (ventral hippocampus, paired-end 91 bp, Illumina) or NextSeq 500 platforms (medial prefrontal cortex, single-end 96 bp; Illumina). Low abundant genes were filtered, keeping genes with at least 1 CPM in at least six samples. With this threshold, we detected 18,947 genes in the medial prefrontal cortex and 19,560 genes in the ventral hippocampus.

Gigliotta A., Trontti K., Väänänen J, & Hovatta I. (2023). Gene expression profiling reveals a role of immune system and inflammation in innate and stress-induced anxiety-like behavior. Frontiers in Genetics, 14, 1173376.

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ChIP-Seq Data Analysis Pipeline

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The purified DNA library was sequenced using an Illumina HighSeq2000 at the Sanford-Burnham Medical Research Institute at Lake Nona, National Genome Library Core Facility. Sequenced regions were aligned to the reference human genome 19 using the Bowtie alignment program that utilizes an extended Burrows-Wheeler indexing for an ultrafast memory efficient alignment [60 (link)]. Peak calling utilized a model-based analysis of ChIP-Seq (MACS) algorithm [61 (link), 62 ]. The overlap analysis, plot of genomic location, sequence extraction, motif identification, and peak filtering were performed using ChIPseek: a web-based analysis for ChIP data [63 (link)]. ChIPseek also employs scripts from BEDtools [64 (link)] using a genome binning algorithm used by the UCSC genome browser to sort genomic regions into groups along the length of chromosome [65 (link)]. Data visualization was carried out using the integrative genomics viewer [66 (link)].

Wilson S., Fan L., Sahgal N., Qi J, & Filipp F.V. (2017). The histone demethylase KDM3A regulates the transcriptional program of the androgen receptor in prostate cancer cells. Oncotarget, 8(18), 30328-30343.

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Small RNA Sequencing of BUNV-infected Cells

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Small RNA sequencing of BUNV-infected Aag2 and U4.4 cells was carried out by Edinburgh Genomics (University of Edinburgh) using the Illumina HighSeq 2000 platform, as previously described [40 (link)]. In short, 2.6 x 10⁶ Aag2 cells/well were seeded into a 6-well plate and left to adhere overnight. Cells were infected with BUNV at a MOI of 10. At 24 hours p.i., RNA was isolated from individual wells using 1 ml TRIzol (Life Technologies) followed by purification, sequencing, analysis and mapping of virus specific small RNAs using viRome [80 (link)]. Small RNA sequences were submitted to the European Nucleotide Archive (accession number PRJEB15203). Data for SBV are referenced under [40 (link)]. The actual number of reads for each experiment is shown S1 Table.

Dietrich I., Shi X., McFarlane M., Watson M., Blomström A.L., Skelton J.K., Kohl A., Elliott R.M, & Schnettler E. (2017). The Antiviral RNAi Response in Vector and Non-vector Cells against Orthobunyaviruses. PLoS Neglected Tropical Diseases, 11(1), e0005272.

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ChIP-Seq Analysis of Transcription Factor Binding

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For ChIP-Seq, 100 μg human cell line chromatin was immunoprecipitated with 5 μg mouse anti-GR antibody. ChIP-Seq libraries were sequenced on an Illumina High-Seq 2000. Sequence reads for each sample were mapped to the hg19 or mm9 genome assemblies as relevant with Bowtie (38 (link)). Duplicate reads were removed, and the remaining unique reads were normalized to 10 million reads. Peak calling was performed by HOMER (39 (link)) using an FDR cutoff of 0.001, a cumulative Poisson p-value of <0.0001, and required a 4-fold enrichment of normalized sequenced reads in the treatment sample over the control/input sample. Normalized sequence reads around each peak were counted in 25 bp bins. Motif discovery was conducted with HOMER package v4.2. In ChIP-Seq data we used the following settings: -size 150 –S 10 –bits (39 (link)). We limited the motif analysis to a 150 bp DNA region around each peak summit. Distance to gene and gene annotations for ChIP-Seq peaks were obtained using GREAT v1.82.

Huffman K.E., Li L.S., Carstens R., Park H., Girard L., Avila K., Wei S., Kollipara R., Timmons B., Sudderth J., Bendris N., Kim J., Villalobos P., Fujimoto J., Schmid S., Deberardinis R.J., Wistuba I., Heymach J., Kittler R., Akbay E.A., Posner B., Wang Y., Lam S., Kliewer S.A., Mangelsdorf D.J, & Minna J.D. (2023). Glucocorticoid mediated inhibition of LKB1 mutant non-small cell lung cancers. Frontiers in Oncology, 13, 1025443.

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RNA-seq Library Preparation and Bioinformatics Analysis

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mRNA was extracted from biopsy samples and used for RNA-Seq library preparation following instructions in the Illumina TruSeq RNA Library Prep Kit v2. Sequencing was run on an Illumina High Seq-2000 in the Mayo Clinic Sequencing Core with 101bp paired end reads. Gene expression counts were obtained using the MAP-RSeq v.2.0.0 workflow (Kalari et al., 2014 (link)) which is part of the Mayo Bioinformatics Core pipeline. MAP-RSeq consists of alignment with TopHat 2.0.12 (Kim et al., 2013 (link)) against the human hg19 genome build and gene counts with the Subread package 1.4.4 (Liao et al., 2019 (link)). Gene annotation files were obtained from Ensemble version 75. Gene counts were normalized using RPKM (Reads Per Kilobase per million Mapped reads). Differential expression analysis was performed using edgeR 2.6.2 (Robinson et al., 2010 (link)). Pathway enrichment analyses were performed using R package RITAN (Rapid Integration of Term Annotation and Network resources, https://www.bioconductor.org/packages/release/bioc/html/RITAN.html).

Mars R.A., Yang Y., Ward T., Houtti M., Priya S., Lekatz H.R., Tang X., Sun Z., Kalari K.R., Korem T., Bhattarai Y., Zheng T., Bar N., Frost G., Johnson A.J., van Treuren W., Han S., Ordog T., Grover M., Sonnenburg J., D’Amato M., Camilleri M., Elinav E., Segal E., Blekhman R., Farrugia G., Swann J.R., Knights D, & Kashyap P.C. (2020). Longitudinal Multi-omics Reveals Subset-Specific Mechanisms Underlying Irritable Bowel Syndrome. Cell, 182(6), 1460-1473.e17.

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Metagenomic DNA Extraction from Neamphius huxleyi

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Three samples of Neamphius huxleyi were used for investigation after washing three times with artificial seawater (ASW) (1.1 g CaCl₂, 10.2 g MgCl₂·6H₂O, 31.6 g NaCl, 0.75 g KCl, 1.0 g Na₂SO₄, 2.4 g Tris-HCl, 0.02 g NaHCO₃, 1 L distilled water, pH 7.6) to eliminate the microbes loosely attached on the sponge surface and canals. Two strategies were used to extract sponge metagenomic DNA: (1) QIAGEN DNeasy Kit (Qiagen) following the manufacturer's protocol; (2) CTAB (Cetyltrimethyl Ammonium Bromide)-based method according to Taylor et al.42 (link). DNA samples extracted by different methods were pooled and mixed thoroughly before deep sequencing.
Deep sequencing was carried out on Genome Analyzer IIx system and Highseq 2000 of Illumina Company using paired-end technology (2 × 120, 2 × 100). Total metagenomic DNA was broken up into 300 bp fragments by Covaris and extracted using QIA quick PCR purification kit (QIAGEN, part # 28104). Adaptors were ligated to the extracted 300 bp fragments. Then, 300 bp fragments were enriched using Phusion DNA Polymerase through low cycle PCR under default conditions with 5 cycles. Cluster generation and sequencing were performed according to the manufacturer's manual.

Li Z.Y., Wang Y.Z., He L.M, & Zheng H.J. (2014). Metabolic profiles of prokaryotic and eukaryotic communities in deep-sea sponge Neamphius huxleyi indicated by metagenomics. Scientific Reports, 4, 3895.

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Sequencing of Drosophila Genomes

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Genomes reported here are derived from two natural population samples collected in Stockholm (59.34°, 17.94°, Sweden) and in Castellana Grotte (40.90°, 17.16°, Bari, Southern Italy) in September and October 2011, respectively (Fig. 1 and Table S2, Supporting Information). Isofemale lines were established from a single wild-caught female and maintained in the laboratory for at least twenty generations at room temperature, and therefore passively inbred, before sequencing. A total of 26 Swedish and 16 Italian strains were individually sequenced. For each strain, DNA was extracted from 30 adult females using Puregene Core Kit A (Qiagen, Germany) according to the manufacturer’s instructions. Short-sequence libraries (~500 bp) were constructed and paired-end sequencing using Illumina High-Seq 2000 platform was performed (100 bp reads). Quality control was performed using FastQC⁷⁶. Raw fastq files were deposited in the Sequence Read Archive (SRA) from the National Center for Biotechnology Information (NCBI) under the BioProject accession: PRJNA390275.

Mateo L., Rech G.E, & González J. (2018). Genome-wide patterns of local adaptation in Western European Drosophila melanogaster natural populations. Scientific Reports, 8, 16143.

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Functional shRNA Screening in Mice

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For shRNA screen procedure, different sets of cells were infected with a pool of approximately 200 lentiviral shRNAs targeting 24 human genes at a representation of ~ 500 cells per shRNAs at Multiplicity of Infection at 1. On day 2 of the post infection, puromycin (Sigma) (1ug/ml) was added to remove any non-infected cells and the selection procedure proceeded for the next 3 days. Afterwards, 100,000 transduced cells were injected into its recipient mice and the Control populations were harvested. For each corresponding samples, shRNA barcodes were PCR-recovered from genomic samples, and analyzed through deep sequencing technology (Illumina High-Seq 2000). Each shRNA read was normalized to its whole population and changes in the relative abundance of each shRNA in the library were measured.

Sa J.K., Yoon Y., Kim M., Kim Y., Cho H.J., Lee J.K., Kim G.S., Han S., Kim W.J., Shin Y.J., Joo K.M., Paddison P.J., Ishitani T., Lee J, & Nam D.H. (2015). In vivo RNAi screen identifies NLK as a negative regulator of mesenchymal activity in glioblastoma. Oncotarget, 6(24), 20145-20159.

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Targeted Gene Sequencing for Rare Diseases

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Most of the patients underwent targeted gene sequencing according to their clinical symptoms. Three patients underwent whole exome sequencing. Genomic DNA of the index patients was enriched for coding exons with Sure Select Human All Exon V6 library preparation Kit and sequenced on an Illumina High Seq 2000. Variants were called using the Cologne Center for Genomics in house analysis pipeline Varbank (https://varbank.ccg.uni-koeln.de/) and filtered according to the expected inheritance model for each patient. Variants and their cosegregation were validated by standard Sanger sequencing.

Yiş U., Becker K., Kurul S.H., Uyanik G., Bayram E., Haliloğlu G., Polat A.İ., Ayanoğlu M., Okur D., Tosun A.F., Serdaroğlu G., Yilmaz S., Topaloğlu H., Anlar B., Cirak S, & Engel A.G. (2017). Genetic Landscape of congenital myasthenic syndromes from Turkey: Novel mutations and clinical insights. Journal of child neurology, 32(8), 759-765.

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