Hiseq 2500 sequencer

Manufactured by Illumina

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The HiSeq 2500 is a high-throughput DNA sequencing system manufactured by Illumina. It is designed to generate large volumes of sequence data rapidly and efficiently. The core function of the HiSeq 2500 is to perform massively parallel sequencing of nucleic acid samples.

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Lab products found in correlation

Agilent 2100 bioanalyzer, by Agilent Technologies (73 mentions) Trizol reagent, by Thermo Fisher Scientific (59 mentions) 2100 bioanalyzer, by Agilent Technologies (48 mentions) Rneasy mini kit, by Qiagen (39 mentions) Bioanalyzer, by Agilent Technologies (32 mentions) Qubit fluorometer, by Thermo Fisher Scientific (28 mentions) Trizol, by Thermo Fisher Scientific (28 mentions) Truseq rna sample prep kit, by Illumina (25 mentions) Kapa library quantification kit, by Roche (18 mentions) Nanodrop 2000, by Thermo Fisher Scientific (18 mentions) Nextera xt dna library preparation kit, by Illumina (16 mentions) Truseq stranded mrna library prep kit, by Illumina (16 mentions) Agilent high sensitivity dna kit, by Agilent Technologies (16 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (16 mentions) Truseq rna sample preparation kit v2, by Illumina (15 mentions) Truseq stranded mrna sample preparation kit, by Illumina (15 mentions) Bioanalyzer 2100 system, by Agilent Technologies (12 mentions) Rneasy kit, by Qiagen (11 mentions) Rneasy micro kit, by Qiagen (11 mentions) Ampure xp bead, by Beckman Coulter (11 mentions)

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HiSeq 2500 (12028 citations) HiSeq sequencer (379 citations) Illumina sequencers (55 citations) HiSeq 2000/2500 sequencer (28 citations) HiSeq 2500 v4 sequencer (15 citations) 2500 sequencer (12 citations) Illumina HiSeq 2500 Sequencer platform (6 citations) HiSeq 2000 or 2500 sequencers (4 citations) HiSeq 2000 and 2500 sequencers (3 citations) HiSeq 2500 1 T sequencer (2 citations)

1 059 protocols using hiseq 2500 sequencer

Comprehensive Multi-Omics Analysis Pipeline

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The DNA/RNA extraction and Whole-exome sequencing and transcriptome analysis were performed as described previously.^{32 (link)} Briefly, genomic DNAs and total RNAs of the tumor and adjacent normal tissue were extracted using the AllPrep DNA/RNA mini kit (Qiagen, Catalog number 80207). Genomic DNAs of peripheral blood were extracted using QIAamp DNA Blood Midi Kit (Qiagen, Catalog number 51183).
Whole-exome libraries were built up as previously described^{33 (link)} and sequenced by 100-bp paired-end reads on HiSeq2500 Sequencer (Illumina, San Diego, CA, USA). Somatic variants (single nucleotide variations (SNVs) and indels) were called using the following parameters, (i) base quality ≥ 15, (ii) sequence depth ≥ 10, (iii) variant depth ≥ 4, (iv) variant frequency in tumor ≥10%, (v) variant frequency in normal < 2%, and (vi) Fisher P-value < 0.05.^{34 (link)} SNVs and indels were annotated based on RefGene using ANNOVAR.^{35 (link)} Transcriptome analysis was performed with TruSeq RNA Library Prep Kit v2 (Illumina, Catalog number 15026495) on HiSeq2500 Sequencer (Illumina, San Diego, CA, USA) according to the manufacturer’s instruction.

Wei T., Leisegang M., Xia M., Kiyotani K., Li N., Zeng C., Deng C., Jiang J., Harada M., Agrawal N., Li L., Qi H., Nakamura Y, & Ren L. (2021). Generation of neoantigen-specific T cells for adoptive cell transfer for treating head and neck squamous cell carcinoma. Oncoimmunology, 10(1), 1929726.

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Illumina Sequencing of Sheared DNA

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Illumina Tight Insert Fragment, 400 bp–2 ug of DNA was sheared to 400 bp using the Covaris LE220 and size selected using the Pippin (Sage Science). The fragments were treated with end-repair, A-tailing and ligation of Illumina compatible adaptors (IDT) using the KAPA-Illumina library creation kit (KAPA Biosystems). The prepared libraries were quantified using KAPA Biosystems’ next-generation sequencing library qPCR kit (Roche) and run on a Roche LightCycler 480 real-time PCR instrument. The quantified libraries were then prepared for sequencing on the Illumina HiSeq sequencing platform using a TruSeq Rapid paired-end cluster kit, v.2, with the HiSeq 2500 sequencer instrument to generate a clustered flowcell for sequencing. Sequencing of the flowcell was performed on the Illumina HiSeq 2500 sequencer using HiSeq Rapid SBS sequencing kits, v.2, following a 2 × 250 indexed run recipe.

Healey A.L., Garsmeur O., Lovell J.T., Shengquiang S., Sreedasyam A., Jenkins J., Plott C.B., Piperidis N., Pompidor N., Llaca V., Metcalfe C.J., Doležel J., Cápal P., Carlson J.W., Hoarau J.Y., Hervouet C., Zini C., Dievart A., Lipzen A., Williams M., Boston L.B., Webber J., Keymanesh K., Tejomurthula S., Rajasekar S., Suchecki R., Furtado A., May G., Parakkal P., Simmons B.A., Barry K., Henry R.J., Grimwood J., Aitken K.S., Schmutz J, & D’Hont A. (2024). The complex polyploid genome architecture of sugarcane. Nature, 628(8009), 804-810.

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Small and mRNA Sequencing Protocol

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For small ncRNA sequencing, total RNA was used as input for sequencing library preparation using QIAseq miRNA Library Kit (Qiagen). Sequence protocol has been previously described [14 (link)]. Briefly, the libraries were sequenced on a HiSeq 2500 sequencer (Illumina, San Diego, CA, USA) using High Output V4 chemistry and single read 100 bases format. The raw sequences were processed into demultiplexed files in compressed fastq format (Illumina). Adaptor sequences and unique molecular identifiers were trimmed from the resulting reads using CLC Genomic Workbench v. 11 (Qiagen), and sequences were mapped to miRBase v. 22 [32 (link)]. Sequence reads counts normalization and differential expression and principal component analyses were performed using DESeq2 [33 (link)].
For mRNA sequencing, total RNA was used as input for sequencing library preparation using TruSeq Stranded mRNA Library Prep (Illumina). The libraries were then sequenced on a HiSeq 2500 sequencer (Illumina) using High Output V4 chemistry. The raw sequence results were processed into demultiplexed files in compressed fastq format (Illumina). Adaptor sequences were trimmed from the resulting reads using CLC Genomic Workbench v. 11 (Qiagen), and sequences were mapped to the canine genome reference CanFam3.1. Sequence reads count normalization and differential expression analysis were performed using DESeq2 [33 (link)].

Yang V.K., Moyer N., Zhou R., Carnevale S.Z., Meola D.M., Robinson S.R., Li G, & Das S. (2024). Defining the Role of the miR-145—KLF4—αSMA Axis in Mitral Valvular Interstitial Cell Activation in Myxomatous Mitral Valve Prolapse Using the Canine Model. International Journal of Molecular Sciences, 25(3), 1468.

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RNA-Seq of Stem Cell Organoids

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For all H9- and ZIP8K8-derived organoids, RNA was purified using an miRNeasy RNA MiniPrep kit (Qiagen). RNA-Seq libraries were generated for H9- and ZIP8K8-derived organoids (Dual SMAD-i (n = 1; five organoids, pooled), Inhibitor-free (n = 1; five organoids, pooled), WNT-i (n = 1; four organoids, pooled), TGFB and WNT-i (n = 1; four organoids, pooled) and Triple-i (n = 1; five organoids, pooled)) using Illumina TruSeq RNA library preparation kits and sequenced on an Illumina HiSeq 2500 sequencer as 100-bp and 76-bp paired-end reads, respectively. For Triple-i ZIP8K8-derived organoids (n = 5; four organoids, pooled), RNA-Seq libraries were generated using a NEBnext UltraDirectional RNA library preparation kit after ribosomal RNA depletion using a NEBNext rRNA depletion kit and sequenced on an Illumina HiSeq 2500 sequencer using 50 cycles of single-end sequencing.

Rosebrock D., Arora S., Mutukula N., Volkman R., Gralinska E., Balaskas A., Aragonés Hernández A., Buschow R., Brändl B., Müller F.J., Arndt P.F., Vingron M, & Elkabetz Y. (2022). Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells. Nature Cell Biology, 24(6), 981-995.

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Single-cell RNA-seq transcriptome profiling

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500 cells per population were sorted into lysis buffer using a BD FACSARIA III or FACSFUSION instruments (BD Biosciences), and cDNA was prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara). Library construction was performed using the NexteraXT DNA sample preparation kit (Illumina) using half the recommended volumes and reagents. Dual-index, single-read sequencing of pooled libraries was run on a HiSeq2500 sequencer (Illumina) with 58-base reads and a target depth of 5 million reads per sample. Base-calling and demultiplexing were performed automatically on BaseSpace (Illumina) to generate FASTQ files.

Klicznik M.M., Morawski P.A., Höllbacher B., Varkhande S.R., Motley S., Kuri-Cervantes L., Goodwin E., Rosenblum M.D., Long S.A., Brachtl G., Duhen T., Betts M.R., Campbell D.J, & Gratz I.K. (2019). Human CD4+CD103+ cutaneous resident memory T cells are found in the circulation of healthy subjects. Science immunology, 4(37), eaav8995.

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Immunoprecipitation and Next-Generation Sequencing

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Immunoprecipitations were carried out as described (Tavares et al. 2012 (link); Latos et al. 2015 (link)) and are detailed in the Supplemental Material. DNA from four immunoprecipitations was pooled for each library generated with the NEB Next DNA library preparation master mix (New England Biolabs, E6040) according to the manufacturer's instructions. Libraries were amplified using 18 PCR cycles, purified using Agencourt AMPure XP SPRI beads (Beckman Coulter, A63881), and size-selected on an agarose gel. DNA was extracted using the QiaQuick gel extraction kit (Qiagen), and its concentration was determined using the KAPA Illumina SYBR Universal Lid Q kit (KAPA Biosystems, KK4824) and Bioanalyzer 2100 system (Agilent). Libraries were sequenced on an Illumina HiSeq 2500 sequencer.

Latos P.A., Sienerth A.R., Murray A., Senner C.E., Muto M., Ikawa M., Oxley D., Burge S., Cox B.J, & Hemberger M. (2015). Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks. Genes & Development, 29(23), 2435-2448.

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Targeted Exome Sequencing of SCN9A, SCN10A, and SCN11A

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The custom capture oligo set was designed by the SureDesign platform from Agilent Technologies (SureSelectXT Custom Capture Oligo) against SCN9A, SCN10A, and SCN11A. This set was specifically designed to capture all exons, the proximal promoter sequence, as well as limited intron sequences and untranslated regions near exons. The target-captured sequencing libraries were constructed using the KAPA LTP Library Preparation Kit (Kapa Biosystems, Inc., Wilmington, MA) combined with a SureSelectXT Reagent Kit (Agilent Technologies), and sequenced on an Illumina Hiseq 2500 sequencer (Illumina, Inc, San Diego, CA) at read length of paired-end 2 × 100 bp. Targeted exome sequencing was performed on an initial cohort of 46 IBD patients that included a mixture of individuals with and without inflammation and abdominal pain. Generated reads were aligned with the GRCh37 human reference genome using the Burrows-Wheeler alignment (20 (link)). Variant detection and analysis were performed using the GATK Best Practice for germline SNP/indel finding workflow (Broad Institute). ANNOVAR software (21 (link)) was used to annotate the variants and identify synonymous, non-synonymous, and deleterious variants for further analysis.

Gonzalez-Lopez E., Imamura Kawasawa Y., Walter V., Zhang L., Koltun W.A., Huang X., Vrana K.E, & Coates M.D. (2018). Homozygosity for the SCN10A Polymorphism rs6795970 Is Associated With Hypoalgesic Inflammatory Bowel Disease Phenotype. Frontiers in Medicine, 5, 324.

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RNA-seq analysis of Cdk8 knockout cells

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RNA was isolated from immortalized BCR-ABL1^p185+Cdk8^fl/fl and Cdk8^Δ/ΔVav-Cre cells. Single-end, 50 bp sequencing of libraries prepared with the Lexogen SENSE mRNA-Seq library preparation kit was performed on an Illumina HiSeq-2500 sequencer. After quality control of raw data with FastQC and removement of adapters and low quality reads with Trimmomatic (version 0.36), reads were mapped to the GENECODE M13 genome using STAR (version 2.5.2b) with default parameters. Then, featureCounts from the Subread package (version 1.5.1) was used to obtain gene counts for union gene models. Differentially expressed (p-adjust < 0.05 and fold change > 2) treatment dependent (Cdk8^fl/fl dimethyl sulfoxide (DMSO) vs. Cdk8^fl/fl Senexin B; Cdk8^Δ/ΔVav-Cre DMSO vs. Cdk8^Δ/ΔVav-Cre Senexin B) and genotype-dependent (Cdk8^fl/fl vs. Cdk8^Δ/ΔVav Cre) genes were identified using DESeq2 (version 1.18.1). For the heatmap, centered and scaled regularized log-transformed^{54 (link)} library size-normalized counts were visualized using the aheatmap function of the R package NMF (version 0.20.6). The command-line version of GSEA was used for GSEA^{55 (link)} and pathway analysis was performed with EnrichR^{56 (link)} using either significant up- or downregulated genes (fold change > 2, padjust < 0.1) of Cdk8^fl/fl vs. Cdk8^Δ/ΔVav Cre cell lines.

Menzl I., Zhang T., Berger-Becvar A., Grausenburger R., Heller G., Prchal-Murphy M., Edlinger L., Knab V.M., Uras I.Z., Grundschober E., Bauer K., Roth M., Skucha A., Liu Y., Hatcher J.M., Liang Y., Kwiatkowski N.P., Fux D., Hoelbl-Kovacic A., Kubicek S., Melo J.V., Valent P., Weichhart T., Grebien F., Zuber J., Gray N.S, & Sexl V. (2019). A kinase-independent role for CDK8 in BCR-ABL1+ leukemia. Nature Communications, 10, 4741.

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iPSC SNV Sequencing Protocol

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The genomic region containing the target SNV site was amplified by PCR in a reaction containing 15 ng of genomic DNA prepared from iPSCs (2500 cells). PCR was performed using Titanium-taq DNA polymerase (Clontech, Palo Alto, CA) under the following conditions: 95 °C for 1 min followed by 32 cycles at 95 °C for 20 s, 68 °C for 30 s, and 72 °C for 30 s. The PCR products were mixed and purified using a MinElute PCR Purification Kit (Qiagen), and then subjected to comprehensive sequencing using a HiSeq 2500 sequencer (101 bp, paired-end, Illumina). The VAF of each SNV was estimated by BWA mapping of the obtained reads. Primer sequences are listed in Supplementary Data 5.

Araki R., Hoki Y., Suga T., Obara C., Sunayama M., Imadome K., Fujita M., Kamimura S., Nakamura M., Wakayama S., Nagy A., Wakayama T, & Abe M. (2020). Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency. Nature Communications, 11, 197.

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mRNA and ncRNA Sequencing in Heart Failure

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For mRNA-Seq, 36 samples were examined (HF, n = 26; and control, n = 10), and for ncRNA-Seq, a total of 50 samples were analyzed (HF, n = 42; and control, n = 8). RNA extraction, determination of purity and integrity of RNA samples, mRNA-Seq, and ncRNA-Seq analysis were performed as previously described by Pérez-Carrillo et al. and Gil-Cayuela et al. [16 (link), 17 (link)]. Briefly, TRIzol^® agent was used to homogenize tissue samples in a TissueLysser LT (Qiagen). RNA extractions were performed using a PureLink™ Kit (Ambion Life Technologies) for mRNA-Seq and the Quik-RNATM miniprep plus kit (Zymo Research) for ncRNA-Seq, according to the manufacturer’s instructions. The cDNA libraries were obtained following Illumina’s recommendations. Sequencing was performed using the SOLiD 5500XL platform for mRNA and the Illumina HiSeq 2500 sequencer for ncRNA.

Delgado-Arija M., Genovés P., Pérez-Carrillo L., González-Torrent I., Giménez-Escamilla I., Martínez-Dolz L., Portolés M., Tarazón E, & Roselló-Lletí E. (2024). Plasma fibroblast activation protein is decreased in acute heart failure despite cardiac tissue upregulation. Journal of Translational Medicine, 22, 124.

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