Hiseq 2000 machine

Manufactured by Illumina

Sourced in United States, China

The HiSeq 2000 is a next-generation sequencing system manufactured by Illumina. It is designed for high-throughput DNA sequencing, capable of generating large volumes of sequence data. The HiSeq 2000 utilizes the Illumina sequencing-by-synthesis technology to perform massively parallel sequencing of DNA samples.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (7 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions) 2100 bioanalyzer, by Agilent Technologies (6 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions) Trizol, by Thermo Fisher Scientific (5 mentions) Ampure xp bead, by Beckman Coulter (4 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions) Rneasy mini kit, by Qiagen (3 mentions) Agilent bioanalyzer 2100 system, by Agilent Technologies (3 mentions) Rneasy rna extraction kit, by Qiagen (3 mentions) Direct zol rna miniprep kit, by Zymo Research (2 mentions) Tri reagent, by Merck Group (2 mentions) Nextera xt kit, by Illumina (2 mentions) Truseq kit, by Illumina (2 mentions) Nebnext dna library prep master mix set for, by Illumina (2 mentions) Bioanalyzer, by Agilent Technologies (2 mentions) Tapestation 2200, by Agilent Technologies (2 mentions) Rneasy, by Qiagen (2 mentions) Truseq stranded rna sample preparation kit, by Illumina (2 mentions) Superscript 3, by Thermo Fisher Scientific (2 mentions)

82 protocols using hiseq 2000 machine

Whole Exome and Transcriptome Sequencing of Tumor Samples

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For 107 patients, we performed WES on tumor DNA and constitutive normal DNA. DNA was extracted using the QIAamp DNA kit (Qiagen) according to manufacturer’s protocol. DNA quantification was performed on a Qubit 2.0 Flourometer (Life Technologies). Libraries for WES were prepared on the SureSelect Automated Library Prep and Capture System (Agilent Technologies) according to the manufacturer’s protocol (version E.3). In brief, genomic DNA (1.5–3 μg) from each sample was fragmented to a length distribution peak of 150 to 200 nt for the preparation of paired-end sequencing libraries. Enrichment for exomic sequence was performed using Agilent SureSelect V4+UTR in-solution capture reagents following vendor’s protocol v2.0.1. Sequencing was carried out on HiSeq 2000 machines (Illumina) with 3 samples multiplexed per lane.
For RNA-Seq, RNA was extracted from 123 patients using the RNA RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. RNA quantification was performed on a Qubit 2.0 Flourometer. Quality was assessed on an Agilent 2100 Bioanalyzer. An RNA integrity number (RIN) of at least 8 was required. RNA-Seq libraries were prepared according to the manufacturer’s protocol (Illumina TruSeq RNA sample preparation v2). Sequencing was performed on Illumina HiSeq 2000 machines with 2–3 samples multiplexed per lane.

Dietrich S., Oleś M., Lu J., Sellner L., Anders S., Velten B., Wu B., Hüllein J., da Silva Liberio M., Walther T., Wagner L., Rabe S., Ghidelli-Disse S., Bantscheff M., Oleś A.K., Słabicki M., Mock A., Oakes C.C., Wang S., Oppermann S., Lukas M., Kim V., Sill M., Benner A., Jauch A., Sutton L.A., Young E., Rosenquist R., Liu X., Jethwa A., Lee K.S., Lewis J., Putzker K., Lutz C., Rossi D., Mokhir A., Oellerich T., Zirlik K., Herling M., Nguyen-Khac F., Plass C., Andersson E., Mustjoki S., von Kalle C., Ho A.D., Hensel M., Dürig J., Ringshausen I., Zapatka M., Huber W, & Zenz T. (2017). Drug-perturbation-based stratification of blood cancer. The Journal of Clinical Investigation, 128(1), 427-445.

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Total RNA Extraction and RNA-seq of Primary GBM

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Total RNA was extracted from 3 primary GBM biopsies (00RTS3 – from the LCRC biospecimen core; and CAURPRVE and H8CPFRSJ – from Bioserve) using Trizol (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Total RNA from sample 00RTS3 was subjected to polyA selection, and the library was prepared using the ScriptSeq Protocol (Epicentre, Madison, WI) and subjected to 2x101 base paired-end sequencing on an Illumina Hi-seq 2000 machine. Total RNA from samples CAURPRVE, H8CPFRSJ, and JY were subjected to ribosomal RNA depletion using the Ribo-Zero kit (Epicentre, Madison, WI) and cDNA libraries were prepared using the Illumina Truseq Stranded Total RNA Sample Prep Kit and subjected to 1x101 base single-end multiplexed sequencing on an Illumina Hi-seq 2000 machine. The RNA-seq data used in this publication is available through GEO Series accession number (in process).

Strong M.J., Blanchard E 4.t.h., Lin Z., Morris C.A., Baddoo M., Taylor C.M., Ware M.L, & Flemington E.K. (2016). A comprehensive next generation sequencing-based virome assessment in brain tissue suggests no major virus - tumor association. Acta Neuropathologica Communications, 4, 71.

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Genomic DNA extraction and sequencing

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Spore colonies from tetrads were independently cultured overnight at 30° in 4 ml of YPD liquid medium. DNA was extracted from each culture using the PrepEase DNA isolation kit from Affymetrix following the manufacturer’s protocol. Genomic DNA fragmentation and library preparation were performed as described previously (Wilkening et al. 2013 (link)). Briefly, 2 μg of genomic DNA was sheared using a Bandelin Sonorex RX 102 sonicating water bath to obtain DNA fragments of 250–500 bp. End repair, dA-tailing, and ligation were done as per the Illumina library preparation protocol with heat inactivation instead of column/magnetic bead- based cleanups. The multiplexed libraries were amplified and size selected for 350–-400 bp using Invitrogen E-Gel (SizeSelect 2%). The size-selected DNA was sequenced (100PE) on Illumina HiSeq 2000 machines at the EMBL Genomics Core Facilities (GeneCore), Heidelberg, Germany. The raw reads obtained were demultiplexed using the FASTX-toolkit (https://github.com/agordon/fastx_toolkit). Demultiplexed reads were processed for quality control (QC) using the NGS QC Toolkit (Patel and Jain 2012 (link)). These QC-filtered high-quality reads were used for further analysis. The sequence data are available from the National Centre for Biotechnology Information Sequence Read Archive under accession no. SRP041856.

Krishnaprasad G.N., Anand M.T., Lin G., Tekkedil M.M., Steinmetz L.M, & Nishant K.T. (2014). Variation in Crossover Frequencies Perturb Crossover Assurance Without Affecting Meiotic Chromosome Segregation in Saccharomyces cerevisiae. Genetics, 199(2), 399-412.

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Chinese Cabbage Genome Sequencing

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Total genomic DNA of the Chinese cabbage ‘CT001’ was extracted from young leaves of a single plant using sodium dodecyl sulfate lysis buffer with a modified version of the firstly described by Dellaporta et al [20 ].
DNA libraries were constructed using a TruSeq DNA PCR-Free Kit (Illumina, San Diego, CA, USA) and library quality control (QC) was performed using the Bioanalyzer DNA ChIP (Agilent Technologies, Santa Clara, CA, USA) to produce short single or paired-end reads on Illumina HiSeq 2000 machines (USA). Illumina paired-end sequencing with 150 bp insert size libraries and Illumina mate pair sequencing with large insert (3 and 5 kb) libraries were performed. Data from short-insert paired-end sequencing and those from mate pair sequencing were combined and the gaps between the neighboring scaffolds were filled with 100 Ns. Raw sequences were deposited in NCBI sequence read archive (SRA) with the accession number SRR9190268, belonging to BioProject accession number PRJNA546028.

Park J.S., Park J.H, & Park Y.D. (2019). Construction of pseudomolecule sequences of Brassica rapa ssp. pekinensis inbred line CT001 and analysis of spontaneous mutations derived via sexual propagation. PLoS ONE, 14(9), e0222283.

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RNA-seq Analysis of Mouse Keratinocytes

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FACS isolated keratinocytes were sorted directly into TRI Reagent (Sigma). Three animals were pooled per condition and all experiments were performed in duplicate. RNA was purified using Direct-zol RNA MiniPrep kit (Zymo Research) per manufacturer’s instructions. Quality of the RNA for sequencing was determined using Agilent 2100 Bioanalyzer, all samples used had RNA integrity numbers (RIN) > 8. Library preparation using Illumina TrueSeq mRNA sample preparation kit was performed at the Weill Cornell Medical College Genomic Core facility, and RNAs were sequenced on Illumina HiSeq 2000 machines. Alignment of reads was done using Tophat with the mm9 build of the mouse genome. Transcript assembly and differential expression was determined using Cufflinks with Refseq mRNAs to guide assembly (Trapnell et al., 2010 (link)). Analysis of RNA-seq data was done using the cummeRbund package in R (Trapnell et al., 2012 (link)). Differentially regulated transcripts were used in Gene Set Enrichment Analysis (GSEA) to find enriched functional GO annotations (Subramanian et al., 2005 (link)). MEME software suite (including TomTom) was used to identify enriched motifs in Skint promoters, the JASPAR vertebrate database was as a source for consensus transcription binding site sequences (Bailey et al., 2009 (link)).

Keyes B.E., Liu S., Asare A., Naik S., Levorse J., Polak L., Lu C.P., Nikolova M., Pasoli H.A, & Fuchs E. (2016). Impaired Epidermal to Dendritic T-Cell Signaling Slows Wound Repair in Aged Skin. Cell, 167(5), 1323-1338.e14.

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RNA-seq Library Preparation and Sequencing

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Total RNA was isolated using TRIzol Reagent (Invitrogen), and the integrity of the total RNA was validated using the Agilent 2100 Bioanalyzer. Libraries were generated following the Illumina protocol for preparing samples for sequencing of mRNA, and 1–10 µg of total RNA was used to build libraries for single-read sequencing on the Illumina Hiseq 2000. mRNA was isolated by polyA selection. The mRNA was then fragmented and randomly primed for reverse transcription, followed by second-strand synthesis to create double-stranded cDNA fragments. Ends of the cDNA fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends. An “A” base was added to the blunt ends followed by ligation to Illumina sequencing adapters. cDNA fragments ranging from 300 to 500 bps were gel purified after the adapter ligation step. PCR amplified cDNA libraries were quantified on the Agilent 2100 Bioanalyzer and diluted to 10 pM for cluster generation and sequencing. Single-end sequencing was performed for 50 cycles by using the Single Read Cluster Generation Kit (TruSeq SR Cluster Kit v3 - cBot –HS, Cat# GD-401-3001) and Sequencing Kit (TruSeq SBS Kit v3– HS, Cat# FC-401-3002) on an Illumina Hiseq 2000 machine. Sequence reads from RNA-sequencing were aligned to genomic sequences.

Bhattacharyya S., Pradhan K., Campbell N., Mazdo J., Vasantkumar A., Maqbool S., Bhagat T.D., Gupta S., Suzuki M., Yu Y., Greally J.M., Steidl U., Bradner J., Dawlaty M., Godley L., Maitra A, & Verma A. (2017). Altered hydroxymethylation is seen at regulatory regions in pancreatic cancer and regulates oncogenic pathways. Genome Research, 27(11), 1830-1842.

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Transcriptional Response to Antifungal Compound

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The C. albicans SC5314 strain was grown overnight as described above, washed with phosphate-buffered saline (PBS), used to inoculate YPD containing 10% fetal bovine serum (FBS) at a 1:30 dilution, and incubated at 37°C for 90 min in the presence or absence of compound 9029936 at 5 μM as previously described (30 (link)). RNA was extracted by using a hot-acid-phenol protocol (78 ). Three biological replicates were obtained for each condition (treated and untreated). To determine the final RNA concentration and quality, samples were analyzed using a 2100 series bioanalyzer (Agilent Technologies, CA).
RNA sequencing was performed at the Genome Sequencing Facility at the Greehey Children’s Cancer Research Institute at the University of Texas Health Science Center at San Antonio. Briefly, cDNA libraries for RNA-seq analysis were prepared from total RNA samples using an Illumina TruSeq stranded mRNA-seq kit. RNA sequencing was performed using an Illumina HiSeq 2000 machine (San Diego, CA) to obtain 100-bp paired-end reads. After the sequencing run, demultiplexing with CASAVA was employed to generate a fastq file for each sample.

Romo J.A., Zhang H., Cai H., Kadosh D., Koehler J.R., Saville S.P., Wang Y, & Lopez-Ribot J.L. (2019). Global Transcriptomic Analysis of the Candida albicans Response to Treatment with a Novel Inhibitor of Filamentation. mSphere, 4(5), e00620-19.

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Single-cell RNA sequencing of MCF10CA spheroids

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For MCF10CA single-cell RNA sequencing experiments, spheroids were dissociated as described above and resuspended in DMEM/F12 medium. Capture, full-length cDNA synthesis and amplification was performed on the C1 Single-Cell Auto Prep system for mRNA Seq (Fluidigm) using the IFC for up to 96 cells (medium size 10–17 µm). Cells at a concentration of 350 cells/µl were mixed with C1 Cell Suspension Reagent (Fluidigm) at a ratio of 4:1 immediately before loading on the IFC. Single-cell capture was assessed with an inverted brightfield microscope. Workflow and reagents for single-cell RNA extraction, reverse transcription (RT) and mRNA amplification (18 cycles) were used as described in the SMARTer Ultra Low RNA Kit (for Fluidigm C1). Sequencing libraries were generated with the Nextera XT kit (Illumina) according to an adapted Fluidigm protocol. Concentration and quality of cDNA and sequencing libraries was assessed by a fluorometer (Qubit) and by electrophoresis (Agilent Bioanalyzer high sensitivity DNA chips). Libraries of up to 24 cells were pooled and sequenced as 1 × 50-bp reads on an Illumina HiSeq 2000 machine.

Tirier S.M., Park J., Preußer F., Amrhein L., Gu Z., Steiger S., Mallm J.P., Krieger T., Waschow M., Eismann B., Gut M., Gut I.G., Rippe K., Schlesner M., Theis F., Fuchs C., Ball C.R., Glimm H., Eils R, & Conrad C. (2019). Pheno-seq – linking visual features and gene expression in 3D cell culture systems. Scientific Reports, 9, 12367.

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Whole-exome Sequencing of Fibroblasts and Leukocytes

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Whole-exome sequencing was performed on DNA extracted from the fibroblasts of P1 and peripheral leukocytes from the parents and P2 and P3 of family A with the Sure Select All Exome V4 capture kit (Agilent) on a HiSeq2000 machine (Illumina). The statistics are provided in Table S1. BWA version 0.5.9 (Li and Durbin, 2009 (link)) and NARWHAL software (Brouwer et al., 2012 (link)) were used to align reads with the reference genome (hg19). Variants were called with GATK (version 1.2–29; McKenna et al., 2010 (link)) and SAMtools (version 0.1.13). Variants were annotated with ANNOVAR software (revision 511; Wang et al., 2010 (link)) and filtered with TIBCO Spotfire version 4.5 (Tables S1 and S2).

Meuwissen M.E., Schot R., Buta S., Oudesluijs G., Tinschert S., Speer S.D., Li Z., van Unen L., Heijsman D., Goldmann T., Lequin M.H., Kros J.M., Stam W., Hermann M., Willemsen R., Brouwer R.W., Van IJcken W.F., Martin-Fernandez M., de Coo I., Dudink J., de Vries F.A., Bertoli Avella A., Prinz M., Crow Y.J., Verheijen F.W., Pellegrini S., Bogunovic D, & Mancini G.M. (2016). Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome. The Journal of Experimental Medicine, 213(7), 1163-1174.

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AGO-PAR-CLIP Profiling of RNA-Binding

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AGO-PAR-CLIP was performed as a modified protocol similar to one previously described [27] (link) using the Millipore 11A9 anti-AGO2 antibody, with one major modification: the Illumina TruSeq kit was used for indexed cDNA library synthesis. Samples were then multiplexed with up to eight samples per lane on an Illumina HiSeq 2000 machine (Supplementary Files 1–2, Supplementary Figure 1, A–C). To define cluster sites, we processed AGO-PAR-CLIP data through PARalyzer [28] (link) and Piranha [29] (link), and clusters were then mapped onto the GC19 transcriptome (Supplementary Figure 1D). We compared unique reads in our datasets with those reported in AGO-CLIP data and found similar percentages of unique reads (Supplementary Figure 1E). Additionally, we found enrichment in the T→C transitions (and complementary C→G transitions) expected in AGO-PAR-CLIP datasets (Supplementary Figure 1F).

Hamilton M.P., Rajapakshe K.I., Bader D.A., Cerne J.Z., Smith E.A., Coarfa C., Hartig S.M, & McGuire S.E. (2016). The Landscape of microRNA Targeting in Prostate Cancer Defined by AGO-PAR-CLIP. Neoplasia (New York, N.Y.), 18(6), 356-370.

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