The cDNA libraries were clustered onto a TruSeq paired-end flow cell and sequenced for 100 bp paired-end reads (2 × 100) using a TruSeq 200 cycle SBS kit (Illumina, San Diego, CA). For the initial run, a phi X 174 (PhiX) bacteriophage genome as well as a universal human reference RNA sample were used as controls on the Illumina HiSeq2000 sequencer (KUMC – Genome Sequencing Facility), and sequenced in parallel with other samples to ensure the data generated for each run are accurately calibrated during the image analysis and data analysis. In addition, the PhiX was spiked into each cDNA sample at approximately 1% as an internal quality control.
Truseq 200 cycle sbs kit
Manufactured by Illumina
Sourced in United States
The TruSeq 200 cycle SBS kit is a sequencing-by-synthesis (SBS) reagent kit designed for use with Illumina sequencing platforms. The kit provides the necessary reagents to perform 200 cycles of sequencing on Illumina instruments.
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Lab products found in correlation
Hiseq 2000 sequencer, by Illumina (2 mentions)
Real time analysis software, by Illumina (1 mentions)
Truseq rna preparation kit, by Illumina (1 mentions)
Hiseq2000 dna sequencer, by Illumina (1 mentions)
Clc genomics workbench 12, by Qiagen (1 mentions)
Ingenuity pathway analysis (ipa), by Qiagen (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
Trizol rna isolation reagent, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna sample preparation kit, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Truseq stranded total rna sample preparation kit, by Illumina (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Nextseq 500, by Illumina (1 mentions)
6 protocols using truseq 200 cycle sbs kit
1
RNA-seq protocol with Illumina HiSeq2000
2
Transcriptome Sequencing of cDNA Libraries
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The validated cDNA libraries were clustered onto a TruSeq paired-end flow cell and subjected to transcriptome sequencing for 100-bp paired-end reads (2 × 100) using a TruSeq 200 cycle SBS kit (Illumina). After the sequencing platform generated sequencing images, pixel-level raw data collection, image analysis, and base calling were performed using Real Time Analysis (RTA) software (Illumina). The base call files were converted to FASTQ files using the CASAVA v.1.8.0 software (Illumina) for downstream analysis.
3
RNA-seq Profiling of Rat Junctional Zone in Hyperglycemic Pregnancy
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Transcript profiles for gd 13.5 rat junctional zone tissue isolated from control and hyperglycemic pregnancies were generated by RNA-seq as previously described.19 (link) cDNA libraries from total RNA samples were prepared with Illumina TruSeq RNA preparation kits (Illumina, San Diego, California, USA). Barcoded cDNA libraries were multiplexed and sequenced with a HiSeq2000 DNA sequencer (100 bp paired-end reads) using a TruSeq 200-cycle SBS kit (Illumina) at the KUMC Genome Sequencing Facility. Reads from *.fastq files were mapped to the rat reference genome (Ensembl Rnor_5.0.78) using CLC Genomics Workbench 12.0 (Qiagen, Redwood City, California, USA). mRNA abundance was expressed in reads per kilobase of exon per million reads mapped. A false discovery rate of 0.05 was used as a cut-off for significant differential expression (euglycemia vs hyperglycemia). Functional patterns of transcript expression were analyzed using Ingenuity Pathway Analysis (Qiagen).
4
Tumor RNA Sequencing Analysis Protocol
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Part of the tumor tissue was resected, and total RNA was extracted using TRIzol® RNA Isolation Reagent (Life Technologies, Carlsbad, CA, USA). Using the RNA extracted from the tumor tissue, an mRNA sequencing library was created using the TruSeq stranded mRNA sample preparation kit (Illumina, San Diego, CA, USA), according to the manufacturer's protocol. The library was subsequently analyzed using an Agilent DNA High Sensitivity Kit (Agilent, Santa Clara, CA, USA) and BioAnalyzer 2100. Ultimately, the library was sequenced on the Illumina HiSeq 2500 platform for subsequent RNA sequencing analysis. To generate a cDNA library from the tumor tissues, clusters of cDNA libraries were created using TruSeq flow cell, and sequences were analyzed using the TruSeq 200 Cycle SBS kit (Illumina), which produced 100-bp end reads. The sequencing results of cDNA libraries generated by Illumina HiSeq 2500 were comparatively analyzed with the information stored in FASTQ format. Gene sets were analyzed using the Functional Annotation Tool from DAVID Bioinformatics Resources 6.7, NIH (http://david.abcc.ncifcrf.gov ) [18 (link)]. Differences in gene expression among the experimental groups were assessed using the fold-change false discovery rate, calculated using reads per kilobase per million mapped reads, with P<0.05 denoting statistically significance.
5
RNA-Seq Library Preparation with TruSeq
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The isolated total RNA was processed for preparing RNA sequencing library using TruSeq stranded total RNA sample preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, rRNAs were depleted from 1 μg of total RNA using rRNA removal beads, followed by enzymatic shearing. After first and second strand cDNA synthesis, A-tailing and end repair were performed for ligation of proprietary primers that incorporate unique sequencing adaptors with index for tracking Illumina reads from multiplexed samples run on a single sequencing lane. For each library, an insert size of approximately 200 bp was confirmed by a bioanalyzer using an Agilent DNA Kit (Agilent Technologies, Santa Clara, CA) and quantification of library was measured by real-time PCR using CFX96 real-time system (BioRad, Hercules, CA, USA). Sequencing of each library was performed on an Illumina NextSeq500 and clusters of the cDNA libraries were generated on a TruSeq flow cell and sequenced for 76-bp paired end reads (2 × 76) with a TruSeq 200 cycle SBS kit (Illumina, San Diego, CA, USA). Raw data were processed, and base calling was performed using the standard Illumina pipeline [CASAVA ver. 1.8.2 (http://support.illumina.com/sequencing/sequencing_software/casava.html ) and RTA ver. 1.18.64].
6
Illumina Paired-End Sequencing Protocol
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Using a TruSeq 200 cycle SBS kit (Illumina), the cDNA libraries were clustered onto a TruSeq paired-end flow cell and sequenced for 100 bp paired-end reads (2×100) using an Illumina HiSeq2000 sequencer (University of Kansas Medical Center Genome Sequencing Facility). To ensure the data generated for each run were accurately calibrated during the image and data analysis, a phi X 174 (PhiX) bacteriophage genome as well as a universal human reference RNA sample were sequenced in parallel with other samples. In addition, the PhiX was spiked into each cDNA sample at approximately 1% as an internal quality control.
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