High-throughput sequencing services were performed at the University of Missouri DNA Core Facility. Libraries were constructed following the manufacturer's protocol with reagents supplied in Illumina's TruSeq mRNA stranded sample preparation kit. Briefly, the polyadenylated mRNA was purified from total RNA and fragmented. Double-stranded cDNA was generated from fragmented RNA, and the index-containing adapters were ligated. The final construct of each purified library was evaluated using the Fragment Analyzer instrument, quantified with the Qubit fluorimeter using the quant-iT HS dsDNA reagent kit (Invitrogen), and diluted according to Illumina's standard sequencing protocol for sequencing on an Illumina HiSeq 2500 sequencer.
Quant it hs dsdna reagent kit
Manufactured by Thermo Fisher Scientific
The Quant-iT HS dsDNA reagent kit is a fluorescence-based reagent designed to quantify double-stranded DNA (dsDNA) samples. The kit includes reagents and solutions necessary to perform sensitive DNA quantification assays.
Automatically generated - may contain errors
Lab products found in correlation
Bioanalyzer 2100, by Thermo Fisher Scientific (3 mentions)
Truseq rna sample preparation kit v2, by Illumina (3 mentions)
Bioanalyzer 2100, by Bio-Rad (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Truseq stranded mrna sample preparation kit, by Illumina (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Truseq stranded mrna library preparation kit, by Illumina (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Nextseq 500, by Illumina (1 mentions)
6 protocols using quant it hs dsdna reagent kit
1
Illumina Sequencing of Fragmented mRNA
2
RNA-seq Analysis of Liver Transcriptome in Liver Disease
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Two micrograms of liver RNA from WD (n = 6) and CTL (n = 6) rats were sent to the University of Missouri’s DNA Core for RNA-seq procedures. Of note, only 6 of the 8 WD rats were used for RNA-seq due to resource constraints. High RNA integrity of each sample was confirmed using the BioAnalyzer 2100 automated electrophoresis system (Bio-Rad, Hercules, CA, USA) prior to cDNA library construction. cDNA library preparation was subsequently performed using the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq RNA sample preparation kit v2. Poly-A containing mRNA was purified from 2 μg of total RNA, RNA was fragmented, double-stranded cDNA was generated from fragmented RNA and the index containing sample identifier adapters were ligated to the ends. The final construct of each purified library was evaluated using the BioAnalyzer 2100 automated electrophoresis system, quantified with the Qubit fluorometer using the quant-iT HS dsDNA reagent kit (Invitrogen, Life Technologies, Grand Island, NY), and diluted according to Illumina’s standard sequencing protocol for sequencing on the HiSeq 2000.
3
RNA-seq Library Preparation and Sequencing
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Total RNA was extracted from 2 separate samples for each ligand treatment using Trizol reagent and further cleaned using the RNAeasy kit from QIAGEN. RNA at a concentration of 100 ng/µL in nuclease-free water was used for library construction. Libraries were constructed following the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq RNA sample preparation kit v2. Briefly, the poly-A containing mRNA was purified from total RNA, RNA was fragmented, double-stranded cDNA was generated from fragmented RNA, and adapters were ligated to the ends.
The final construct of each purified library was evaluated using the BioAnalyzer 2100 automated electrophoresis system, quantified with the Qubit fluorometer using the quant-iT HS dsDNA reagent kit (Invitrogen), and diluted according to Illumina’s standard sequencing protocol for sequencing on the HiSeq 2000. The constructed libraries were loaded onto a standard 7-lane flow cell. Samples were sequenced using an Illumina HiSeq 2000 platform utilizing 100 single base reads and multiplexed so that ~20 million reads per sample could be achieved.
The final construct of each purified library was evaluated using the BioAnalyzer 2100 automated electrophoresis system, quantified with the Qubit fluorometer using the quant-iT HS dsDNA reagent kit (Invitrogen), and diluted according to Illumina’s standard sequencing protocol for sequencing on the HiSeq 2000. The constructed libraries were loaded onto a standard 7-lane flow cell. Samples were sequenced using an Illumina HiSeq 2000 platform utilizing 100 single base reads and multiplexed so that ~20 million reads per sample could be achieved.
4
Bacterial 16S Amplicon Sequencing
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The University of Missouri DNA Core Facility prepared bacterial 16S ribosomal DNA amplicons from extracted fecal DNA by amplification of the V4 hypervariable region of the 16S rDNA as we have previously described77 –79 (link). Final amplicon pool was evaluated using the Advanced Analytical Fragment Analyzer automated electrophoresis system, quantified with the Qubit flourometer using the quant-iT HS dsDNA reagent kit (Invitrogen), and diluted according to Illumina’s standard protocol for sequencing on the MiSeq. Sequence data was generated using a paired-end, 250 base pair read length.
5
Illumina TruSeq mRNA Library Preparation
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Libraries were constructed following the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq mRNA Stranded Library Preparation Kit. Briefly, the poly-A containing mRNA was purified from total RNA, mRNA was fragmented, double-stranded cDNA generated from fragmented RNA, and the index containing adapters were ligated to the ends.
The final construct of each purified library was evaluated using the Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) automated electrophoresis system, quantified with the Qubit flourometer using the quant-iT HS dsDNA reagent kit (Invitrogen), and diluted according to Illumina’s standard sequencing protocol for sequencing on the NextSeq 500. Libraries were sequenced at the University of Missouri DNA Core Facility to obtain 75 base pair, single end reads.
The final construct of each purified library was evaluated using the Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) automated electrophoresis system, quantified with the Qubit flourometer using the quant-iT HS dsDNA reagent kit (Invitrogen), and diluted according to Illumina’s standard sequencing protocol for sequencing on the NextSeq 500. Libraries were sequenced at the University of Missouri DNA Core Facility to obtain 75 base pair, single end reads.
6
RNA-seq Library Preparation from Liver Samples
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Two micrograms of liver RNA were sent to the University of Missouri’s DNA Core for RNA-seq procedures. Briefly, high RNA integrity of each sample was confirmed using the BioAnalyzer 2100 automated electrophoresis system (Bio-Rad, Hercules, CA, USA) prior to cDNA library construction. cDNA library preparation was subsequently performed using the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq RNA sample preparation kit v2. Briefly, poly-A containing mRNA was purified from 2 μg of total RNA, RNA was fragmented, double-stranded cDNA was generated from fragmented RNA and the index containing sample identifier adapters were ligated to the ends. The final construct of each purified library was evaluated using the BioAnalyzer 2100 automated electrophoresis system, quantified with the Qubit fluorometer using the quant-iT HS dsDNA reagent kit (Invitrogen, Life Technologies, Grand Island, NY), and diluted according to Illumina’s standard sequencing protocol for sequencing on the HiSeq 2000.
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