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Hiseq 2000 sequencing machine

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The HiSeq 2000 is a high-throughput DNA sequencing platform designed by Illumina. It utilizes sequencing-by-synthesis technology to generate large volumes of sequence data. The HiSeq 2000 is capable of producing up to 600 gigabases of sequence data per run.

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Lab products found in correlation

Nextflex directional rna seq kit, by PSC Biotech (2 mentions) Truseq rna sample preparation protocol, by Illumina (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Spectrum plant total rna kit, by Merck Group (1 mentions) Truseq rna sample preparation kit, by Illumina (1 mentions) Ribo zero rrna removal kit, by Illumina (1 mentions) Nanodroptm 1000 spectrometer, by Thermo Fisher Scientific (1 mentions) Rnase free dnase set, by Qiagen (1 mentions) Trizol ls, by Thermo Fisher Scientific (1 mentions) Illuminatruseq stranded mrna sample prep kits, by Illumina (1 mentions) Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) 7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions) Direct zol rna miniprep kit, by Zymo Research (1 mentions) Epoch 2 microplate spectrophotometer, by Agilent Technologies (1 mentions) Fastx toolkit, by Illumina (1 mentions) Qiaquick pcr extraction kit, by Qiagen (1 mentions) Scriptseq mrna seq library preparation kit, by Illumina (1 mentions) Truseq rnaseq sample prep kit, by Illumina (1 mentions) Rneasy kit, by Qiagen (1 mentions)

15 protocols using hiseq 2000 sequencing machine

1

Illumina Strand-specific RNA-seq Protocol

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Strand-specific RNA-seq libraries were prepared using the dUTP-based, Illumina-compatible NEXTflex Directional RNA-Seq Kit (Bioo Scientific) according to the manufacturer’s directions. All libraries were sequenced with an Illumina HiSeq 2000 sequencing machine.
Beyaz S., Mana M.D., Roper J., Kedrin D., Saadatpour A., Hong S.J., Bauer-Rowe K.E., Xifaras M.E., Akkad A., Arias E., Pinello L., Katz Y., Shinagare S., Abu-Remaileh M., Mihaylova M.M., Lamming D.W., Dogum R., Guo G., Bell G.W., Selig M., Nielsen G.P., Gupta N., Ferrone C.R., Deshpande V., Yuan G.C., Orkin S.H., Sabatini D.M, & Yilmaz Ö.H. (2016). High fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature, 531(7592), 53-58.
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2

Transcriptional Response to Flg22 Elicitation

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Total RNA was extracted from the wild-type and bsu-q seedlings treated with flg22 or mock solution using the Spectrum Plant Total RNA kit (Sigma). cDNA libraries were constructed using a TruSeq RNA sample preparation kit (Illumina) according to the manufacturer’s instructions. Samples of three replicates were sequenced on an Illumina HiSEQ2000 sequencing machine. Mapping of reads to the reference genome (TAIR10) and calling differentially expressed genes was conducted via the iPlant collaborative Discovery Environment (de.iplantcollaborative.org/de/)36 . Expression data was clustered and visualized with the TIGR MeV program (http://mev.tm4.org/)37 (link). Scatter plots of flg22-responsive genes were analyzed with Microsoft Excel. GO analysis was performed with agriGO v2.0 (www.http://systemsbiology.cau.edu.cn/agriGOv2/index.php) using default setting38 (link).
Park C.H., Bi Y., Youn J.H., Kim S.H., Kim J.G., Xu N.Y., Shrestha R., Burlingame A.L., Xu S.L., Mudgett M.B., Kim S.K., Kim T.W, & Wang Z.Y. (2022). Deconvoluting signals downstream of growth and immune receptor kinases by phosphocodes of the BSU1 family phosphatases. Nature plants, 8(6), 646-655.
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3

Directional RNA-seq Library Preparation

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For single-end library construction: Total RNA was depleted of rRNA using the Ribo-Zero rRNA Removal Kit (Epicentre). RNA libraries were prepared using the dUTP-based, Illumina-compatible NEXTflex Directional RN-Seq Kit (Bioo Scientific). For paired-end library construction (performed by BGI), total RNA was enriched for poly(A) mRNA with oligo(dT) beads and treated with buffer to yield 200–700 nt fragments. First-strand cDNA was synthesized using random hexamer primers; second-strand cDNA was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I. cDNA was run on an Agarose gel for suitable fragment size selection, followed by a purification, adaptor ligation, and PCR amplification. All libraries (both single-end and paired-end) were sequenced using an Illumina HiSeq 2000 sequencing machine.
Denzler R., Agarwal V., Stefano J., Bartel D.P, & Stoffel M. (2014). Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. Molecular cell, 54(5), 766-776.
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4

RNA Isolation and Sequencing from Tumor Tissues

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RNA isolations were performed using the RNeasyH Midi Kit (QIAGEN Inc., Valencia, CA, USA), following the protocol for isolating cytoplasmic RNA. Briefly, tumor tissues were processed and the centrifugation steps were performed at 2,850 ×g. DNA was removed using the RNase-Free DNase Set (QIAGEN Inc.) at the recommended step in the RNeasyH protocol. RNA concentration was determined using a NanoDropTM 1000 Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The sequencing library was constructed according to Illumina’s TruSeq RNA Sample Preparation Protocol.12 (link) After normalization, the DNA sample libraries were pooled into four libraries, and the pooled libraries were sequenced on an Illumina HiSeq 2000 sequencing machine (Illumina Inc., San Diego, CA, USA).
Ji F., Liu X., Wu Y., Fang X, & Huang G. (2015). Overexpression of PI3K p110α contributes to acquired resistance to MET inhibitor, in MET-amplified SNU-5 gastric xenografts. Drug Design, Development and Therapy, 9, 5697-5704.
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5

Isolation and RNA-seq of Hair Follicle, Epidermis, and Wound-Activated Stem Cells

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Normal HF-, Epd- and SCC-SCs were isolated as previously described (Ge et al., 2016 (link)). For wound-activated SCs (Wd-SCs), adult second telogen (P60) Sox9CreER; R26YFP female mice were subjected to full-thickness 7mm punch wounds. Seven days post-wounding, single cell suspensions were obtained by FACS as Lineage-YFP+ Integrin-α6+ (HF-derived) and Lineage-YFPneg Integrin-α6+ (Epd-derived). Cells were lysed with TrizolLS (Invitrogen) and total RNA was isolated with the Direct-zol RNA MiniPrep kit (Zymo Research) and submitted to the Genomics Resources Core Facility of the Weill Cornell Medical College for quality control (determined using Agilent 2100 Bioanalyzer, with all samples passing the quality threshold of RNA integrity numbers (RIN 8). Library constructions were performed using IlluminaTruSeq Stranded mRNA Sample Prep Kits, and sequencing was performed on an Illumina HiSeq2000 sequencing machine.
For RNA quantitative PCR, primers DNA oligos were synthesized from Eurofinsgenomics complementary DNAs were generated from 1μg of total RNA using the SuperScript Vilo cDNA synthesis kit (Life Tech), diluted and used as templates for real-time PCR performed with the 7900HT Fast Real-Time PCR System (Applied Biosystems) and gene-specific primers listed in Table S2. Glyceraldehyde dehydrogenase (GAPDH) was used as a loading reference.
Ge Y., Gomez N.C., Adam R.C., Nikolova M., Yang H., Verma A., Lu C.P., Polak L., Yuan S., Elemento O, & Fuchs E. (2017). Stem Cell Lineage Infidelity Drives Wound-Repair and Cancer. Cell, 169(4), 636-650.e14.
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6

RNA-Seq Analysis of C. elegans

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Purified RNA was quantified and assessed for quality using an Epoch 2 microplate spectrophotometer (BioTek). cDNA libraries were prepared from 500 ng RNA per sample. Libraries were then sequenced on an Illumina HiSeq 2000 sequencing machine using paired-end sequencing with a read length of 100 nucleotides. Adaptor sequences and low-quality reads were removed using Trimmomatic, and the RNA-Seq reads were then mapped to the C. elegans genome using STAR 2.5.3a with default settings. Transcript abundance, measured as read counts per gene, was extracted using HTSeq. Differential gene expression analysis was performed using GFold34 (link). Gene ontology (GO) analysis was conducted using DAVID.
Feng M., Gao B., Ruiz D., Garcia L.R, & Sun Q. (2024). Bacterial vitamin B6 is required for post-embryonic development in C. elegans. Communications Biology, 7, 367.
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7

Transcriptome Profiling of Tumor and Normal Tissues

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Total RNA was isolated from frozen tumor and matched normal tissues using the reagent Trizol (Invitrogen). The sequencing library was constructed according to Illumina's TruSeq RNA Sample Preparation Protocol. Poly-A containing mRNA was purified from total RNA using magnetic beads with oligo-dT, followed by fragmentation. First-strand cDNA was synthesized using random hexamers and reverse transcriptase. Second-strand cDNA was synthesized with high quality deoxyribonucleotide triphosphates (dNTPs), ribonuclease H (RNase H), and DNA polymerase. Then the new double-strand cDNA was end-repaired and a single nucleotide “A” was added. Different in-house designed 6 bp adapters were ligated to the corresponding samples. DNA fragments with selected size and adapters were purified and amplified by PCR. After normalization, the DNA sample libraries were pooled into 4 libraries, and the pooled libraries were sequenced on an Illumina HiSeq 2000 sequencing machine.
Xiong Z., Yu H., Ding Y., Feng C., Wei H., Tao S., Huang D., Zheng S.L., Sun J., Xu J, & Fang Z. (2014). RNA Sequencing Reveals Upregulation of RUNX1-RUNX1T1 Gene Signatures in Clear Cell Renal Cell Carcinoma. BioMed Research International, 2014, 450621.
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8

Transcriptomic Response of N2 and npr-1 Worms to Pathogens

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N2 and npr-1(ur89) worms were exposed to either PA14, BT B-18247 or OP50 for either 12 or 24 h of exposure. The experiment had 3 replicates of each treatment combination (a total of 36 samples across treatment combinations and replicates). Exposure experiments were performed on large Agar plates (15 cm diameter), which were fully covered with a bacterial lawn, thus minimizing escape responses and resulting in comparable occupancy rates across the treatment combinations. At both time points (12 & 24), worms were washed off the exposure plates with PBS containing 0.3 % Tween20® and resuspended in TRIzol® (Life Technologies) reagent. Prior to RNA extraction, worm suspensions were treated five times with a freeze-thaw cycle using liquid nitrogen and a thermo block at 45 °C. RNA extraction was performed using a NucleoSpin® mRNA extraction kit (Macherey-Nagel). RNA samples were treated with DNAse, and then stored at –80 °C. RNA libraries were prepared for sequencing using standard Illumina protocols. Libraries were sequenced on an Illumina HiSeq™ 2000 sequencing machine with a paired-end strategy and read length of 100 nucleotides.
Nakad R., Snoek L.B., Yang W., Ellendt S., Schneider F., Mohr T.G., Rösingh L., Masche A.C., Rosenstiel P.C., Dierking K., Kammenga J.E, & Schulenburg H. (2016). Contrasting invertebrate immune defense behaviors caused by a single gene, the Caenorhabditis elegans neuropeptide receptor gene npr-1. BMC Genomics, 17, 280.
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9

Hth-Exd-Ubx Homeodomain SELEX Experiment

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The Hth-Exd-Ubx SELEX experiment was carried out following previously published methods8 (link),61 . In brief, after expressing and purifying the wild-type homeodomain proteins, a final concentration of 50 nM was assembled, incubated with excess DNA (10–20 fold) for 30 minutes and loaded onto an EMSA gel. A DNA library with 30 randomized bases was used. The TF-bound fraction was isolated from the gel and amplified and either subjected to another round of enrichment or prepared for sequencing. Three rounds of enrichment were performed. After each selection round, the DNA was extracted from the gel and amplified by using Ilumina’s small RNA primer sets. Sequencing barcodes were added in a five-cycle PCR step, and the final library was gel-purified using a native TBE gel before sequencing. Libraries were sequenced at the New York Genome Center using separate lanes on an Illumina HiSeq 2000 sequencing machine.
Rube H.T., Rastogi C., Feng S., Kribelbauer J.F., Li A., Becerra B., Melo L.A., Do B.V., Li X., Adam H.H., Shah N.H., Mann R.S, & Bussemaker H.J. (2022). Prediction of protein–ligand binding affinity from sequencing data with interpretable machine learning. Nature Biotechnology, 40(10), 1520-1527.
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10

High-throughput sequencing of Hth-Exd complexes

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Libraries for HthFL-Exd and HthFL-ExdR2A,R5A (Lib-16) were sequenced using a v2 75-cycle high-output kit on an Illumina NEXTSeq Series desktop sequencer at the Genome Center at Columbia University. Libraries Lib-Hth-F and Lib-Hth-R with either Hth or Exd shape-readout mutant in complex with the respective other wild-type protein and Dfd, as well as the Lib-30 HthFL-Exd-Dfd experiment were all sequenced at the New York Genome Center using separate lanes on an Illumina HiSeq 2000 sequencing machine. Libraries Lib-Hth-F and Lib-Hth-R with wild-type proteins were also sequenced on a HiSeq instrument at a different facility. Libraries were trimmed to remove Illumina- and library-internal adapter sequences using the FASTX toolkit (Hanon lab) and loaded into the R environment using the R package named SELEX (http://bioconductor.org/packages/SELEX) (Riley, 2014 (link)).
Kribelbauer J.F., Loker R.E., Feng S., Rastogi C., Abe N., Rube H.T., Bussemaker H.J, & Mann R.S. (2020). Context-dependent gene regulation by homeodomain transcription factor complexes revealed by shape-readout deficient proteins. Molecular cell, 78(1), 152-167.e11.
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