Nextseq 500 sequencing instrument

Manufactured by Illumina

Sourced in United States

The NextSeq 500 is a high-throughput DNA sequencing instrument designed for a wide range of applications, including gene expression profiling, whole-genome sequencing, and targeted sequencing. It utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequencing data. The NextSeq 500 is capable of producing up to 120 gigabases of sequencing data per run, with read lengths up to 150 base pairs.

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Lab products found in correlation

Flexigene dna kit, by Qiagen (3 mentions) Ribo zero gold, by Illumina (2 mentions) Rneasy mini rna isolation kit, by Qiagen (2 mentions) Truseq stranded total rna prep kit, by Illumina (2 mentions) Rnalater, by Thermo Fisher Scientific (2 mentions) Tri reagent protocol, by Merck Group (1 mentions) Facsaria 3, by BD (1 mentions) Agilent 2200 tapestation assay, by Agilent Technologies (1 mentions) Truseq rna sample preparation kit, by Illumina (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) Moflo cell sorter, by Beckman Coulter (1 mentions) Quant it ribogreen rna reagent, by Thermo Fisher Scientific (1 mentions) Basespace sequencing hub, by Illumina (1 mentions) Kapa library quant kit, by Roche (1 mentions) Nextseq 500 high output kit, by Illumina (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Rneasy micro kit, by Qiagen (1 mentions) Qiaseq upx 3 transcriptome kit, by Qiagen (1 mentions) Rneasy kit, by Qiagen (1 mentions) Tapestation 4200, by Agilent Technologies (1 mentions)

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14 protocols using nextseq 500 sequencing instrument

Lentivirus Guide Sequence Analysis

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Example 4

Genomic DNA was extracted from cell pellets of 100 million cells using a QIAmp genomic DNA blood kit (Qiagen). 100 mg of purified genomic DNA was PCR amplified using Taq DNA polymerase and primers binding the lentivirus upstream and downstream of the guide-tracr-SENSR™ sequence. Sequencing was performed on a NextSeq500 sequencing instrument (Illumina) with a 150-cycle paired-end DNA sequencing kit to read both the sequence of the guide and the SENSR™ sequence.

US11078483B1. Methods for measuring and improving CRISPR reagent function (2021-08-03). KSQ Therapeutics, Inc. [US]. Inventors: Gregory V. Kryukov [US], Michael R. Schlabach [US], Jason J. Merkin [US].

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RNA-Seq of Sorted Microglia and Brain

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Percoll-enriched microglia were sorted using a Becton-Dickinson FACSAria III cell sorter at the OSUCCC Analytical Cytometry facility. Microglia were identified by CD11b⁺/CD45^low expression. Cells were pelleted and lysed in Arcturus PicoPure Extraction Buffer immediately after sorting. The Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) was used to purify and concentrate total RNA from sorted microglia. During RNA isolation, samples were treated with on-column DNase digestion for 15 min at 23 °C to eliminate contaminating genomic DNA. A 1-mm coronal brain section was also collected from each brain and snap frozen in liquid nitrogen. RNA was isolated using the Tri-Reagent protocol (Sigma-Aldrich). RNA quality and integrity was determined using the Agilent 2200 TapeStation assay (Agilent Technologies). RNA-Seq was performed on sorted microglia and brain section (Bregma - 1.5 mm) RNA at the Hussman Institute for Human Genomics Sequencing Core Facility (University of Miami, Miami, FL). Briefly, RNA-Seq libraries were prepared using the Ovation SoLo RNA-Seq System with AnyDeplete rRNA to remove rRNA and other abundant transcripts according to the manufacturer’s recommendation (Nugen). RNA-Seq libraries were run on an Illumina NextSeq 500 sequencing instrument according to the protocols described by the manufacturer.

O’Neil S.M., Witcher K.G., McKim D.B, & Godbout J.P. (2018). Forced turnover of aged microglia induces an intermediate phenotype but does not rebalance CNS environmental cues driving priming to immune challenge. Acta Neuropathologica Communications, 6, 129.

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Thymocyte Isolation and RNA-Seq

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Thymocytes were prepared from three mice of each genotype (WT, Hoil^ΔCd4 and Sharpin^cpdm), and CD69⁺ MHC I^low and CD69⁺ MHC I^high cells were FACS purified on a MoFlo cell sorter (Beckman Coulter), with a dump channel to gate out PI⁺, CD25⁺, CD44⁺, NK1.1⁺, B220⁺, MHC II⁺, Gr1⁺, Mac-1⁺ and δTCR⁺ cells. Sorted cells were preserved in RNAlater (ThermoFisher Scientific) and frozen at −80 °C, then RNA was isolated with RNeasyPlus Mini kit (Qiagen). Messenger RNA reverse transcription and complementary DNA libraries were prepared using the TruSeq RNA Sample preparation kit (Illumina) following the manufacturer's instructions. Indexed sample libraries were subjected to 75 base single-end sequencing using the 75 cycle high-output kit v2 chemistry for the NextSeq 500 sequencing instrument (Illumina).

Teh C.E., Lalaoui N., Jain R., Policheni A.N., Heinlein M., Alvarez-Diaz S., Sheridan J.M., Rieser E., Deuser S., Darding M., Koay H.F., Hu Y., Kupresanin F., O'Reilly L.A., Godfrey D.I., Smyth G.K., Bouillet P., Strasser A., Walczak H., Silke J, & Gray D.H. (2016). Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis. Nature Communications, 7, 13353.

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Quantitative Analysis of Liver RNA

Cited 1 time

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Liver RNA was purified and subjected to quantitative PCR analysis (supplementary material)^{50 (link)}.
Psd3 mRNA was quantitated using the primer probe set Mm01351099_m1 (ThermoFisher Scientific). RNA transcript levels were normalized to total RNA levels using Quant-iT RiboGreen RNA reagent (ThermoFisher Scientific)^{51 (link)}.
For lipogenic and inflammatory gene expression, total RNA was sequenced on a NextSeq500 sequencing instrument (Illumina) (supplementary material).

Mancina R.M., Sasidharan K., Lindblom A., Wei Y., Ciociola E., Jamialahmadi O., Pingitore P., Andréasson A.C., Pellegrini G., Baselli G., Männistö V., Pihlajamäki J., Kärjä V., Grimaudo S., Marini I., Maggioni M., Becattini B., Tavaglione F., Dix C., Castaldo M., Klein S., Perelis M., Pattou F., Thuillier D., Raverdy V., Dongiovanni P., Fracanzani A.L., Stickel F., Hampe J., Buch S., Luukkonen P.K., Prati D., Yki-Järvinen H., Petta S., Xing C., Schafmayer C., Aigner E., Datz C., Lee R.G., Valenti L., Lindén D, & Romeo S. (2022). PSD3 downregulation confers protection against fatty liver disease. Nature Metabolism, 4(1), 60-75.

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miRNA Library Preparation and Sequencing

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We used 10 ng of size-selected ribosome-protected RNA (17–37 nt) as the starting material for the QIAseq miRNA Library Prep Kit (cat #331502). RNA fragments were ligated to adapters at the 3′ and 5′ ends, reverse transcribed, and amplified. The resulting libraries were size selected for 185–191 bp fragments. The libraries were quantitated by qPCR using the Applied Biosystems ViiA7 qPCR instrument and a KAPA Library Quant Kit (p/n KK4824). All samples were pooled equimolarly and sequenced on a NextSeq 500 High Output v2.5 flowcell (Illumina p/n 20024906) using the Illumina NextSeq 500 sequencing instrument with a single-read configuration (75 bp). An average of 42 million reads per sample was sequenced. FastQ file generation was executed using Illumina’s cloud-based informatics platform, BaseSpace Sequencing Hub.

Shaiken T.E., Grimm S.L., Siam M., Williams A., Rezaeian A.H., Kraushaar D., Ricco E., Robertson M.J., Coarfa C., Jain A., Malovannaya A., Stossi F., Opekun A.R., Price A.P, & Dubrulle J. (2023). Transcriptome, proteome, and protein synthesis within the intracellular cytomatrix. iScience, 26(2), 105965.

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Whole Exome Sequencing for Variant Identification

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Blood samples of the probands and their parents were collected for identifying the source of variants. Genomic DNAs were extracted from blood samples using the Qiagen Flexi Gene DNA kit (Qiagen, Hilden, Germany). WES was performed using a NextSeq500 sequencing instrument (Illumina, San Diego, California, United States) following previously described standard procedures (Wang et al., 2018 (link); Luo et al., 2022 (link)). The sequencing data were generated by massively parallel sequencing with an average depth of >125x and >98% coverage of the capture region on the chip for obtaining high-quality reads that were mapped to the Genome Reference Consortium Human genome build 37 by Burrows-Wheeler alignment. Single-nucleotide point variants and indels were called with the Genome Analysis Toolkit.

Yan H.J., He Y.Y., Jin L., Guo Q., Zhou J.H, & Luo S. (2023). Expanding the phenotypic spectrum of KCNK4: From syndromic neurodevelopmental disorder to rolandic epilepsy. Frontiers in Molecular Neuroscience, 15, 1081097.

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RNA-Seq Analysis of 70 MB Cancers

Cited 2 times

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RNA-Seq was performed in this cohort of 70 MB cases as described in a previous study [8 (link)]. Briefly, RNA-Seq was run in a Nextseq 500 sequencing instrument (Illumina) for paired-end reads. The RNA-Seq data of this cohort are available in Gene Expression Omnibus (GSE143940 and GSE158413). RNA-Seq raw data were aligned by Kallisto [67 (link)] and the gene expression table was extracted by the tximport [68 (link)] package in R environment. For clustering, unsupervised clustering analysis was performed based on the 10,000 most differentially expressed genes using the consensus clustering default parameters by Rtsne and validated by 22 subgroup-specific signature gene expression levels [69 (link)]. RNA-Seq results were sent to Taylor’s laboratory at the Hospital for Sick Children, Toronto, to help with counterpart clustering.

Wu K.S., Jian T.Y., Sung S.Y., Hsieh C.L., Huang M.H., Fang C.L., Wong T.T, & Lin Y.L. (2022). Enrichment of Tumor-Infiltrating B Cells in Group 4 Medulloblastoma in Children. International Journal of Molecular Sciences, 23(9), 5287.

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Comprehensive Transcriptome Analysis of Jak2VF+ Mouse Cells

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LSK and LK cells were sorted from Jak2^VF/+ mice treated with PBS or IL-1β at 16 weeks after treatment using a FACS Aria II. Total RNA was extracted from LSK and LK cells using RNeasy micro kit (Qiagen). BM MSCs were treated with PBS or IL-1β for 72 h and total RNA was extracted using RNeasy Mini kit (Qiagen). RNA sequencing was performed using NextSeq 500 High Output Kit and NextSeq 500 sequencing instrument (Illumina). RNA-seq data alignment was performed using UCSC mm10 reference genome with HISAT2 V2.0.1. The read counts and differential analysis were done using Genomic Alignments and DESeq2. P-adjusted value of <0.05 with a log₂fc > 0.5 was considered as significant change in gene expression.

Rahman M.F., Yang Y., Le B.T., Dutta A., Posyniak J., Faughnan P., Sayem M.A., Aguilera N.S, & Mohi G. (2022). Interleukin-1 contributes to clonal expansion and progression of bone marrow fibrosis in JAK2V617F-induced myeloproliferative neoplasm. Nature Communications, 13, 5347.

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Transcriptome Analysis of Duodenal Biopsies

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Total RNA was extracted from the same duodenal PaxFPE biopsy specimens using further cuttings from where histomorphometry was to be measured and counted as previously described.²⁴ (link) For the extraction, an RNeasy Kit (Qiagen, Hilden, Germany) was used according to the manufacturer’s instructions. Library preparation and next-generation sequencing (NGS) was performed by Qiagen NGS Service. The library preparation was done using the QIAseq UPX 3′ Transcriptome Kit (Qiagen). A total of 10 ng of purified RNA was converted into complementary DNA NGS libraries, and a quality check was performed using TapeStation 4200 (Agilent, Santa Clara, CA) or Agilent Bioanalyzer (Agilent). Sequencing was performed on a NextSeq500 sequencing instrument according to the manufacturer’s instructions (Illumina, San Diego, CA).

Dotsenko V., Oittinen M., Taavela J., Popp A., Peräaho M., Staff S., Sarin J., Leon F., Isola J., Mäki M, & Viiri K. (2020). Genome-Wide Transcriptomic Analysis of Intestinal Mucosa in Celiac Disease Patients on a Gluten-Free Diet and Postgluten Challenge. Cellular and Molecular Gastroenterology and Hepatology, 11(1), 13-32.

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Whole-Exome Sequencing for Genetic Variants

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Blood samples of the probands, their parents, and other available family members were collected. Genomic DNAs were extracted from blood samples using the Qiagen Flexi Gene DNA kit (Qiagen, Hilden, Germany). WES was performed using a NextSeq500 sequencing instrument (Illumina, San Diego, California, USA) following the standard procedures previously described (Wang et al., 2018 (link)). The sequencing data were generated by massively parallel sequencing with an average depth of >125x and >98% coverage of the capture region on the chip for obtaining high-quality reads that were mapped to the Genome Reference Consortium Human genome build 37 by Burrows-Wheeler alignment. Variants were called and qualified with the Genome Analysis Toolkit (DePristo et al., 2011 (link)). Sanger sequences were used to validate candidate variants.

Luo S., Ye X.G., Jin L., Li H., He Y.Y., Guan B.Z., Gao L.D., Liang X.Y., Wang P.Y., Lu X.G., Yan H.J., Li B.M., Chen Y.J, & Liu Z.G. (2023). SZT2 variants associated with partial epilepsy or epileptic encephalopathy and the genotype-phenotype correlation. Frontiers in Molecular Neuroscience, 16, 1162408.

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