Whole mRNA transcriptome library preparation and sequencing was performed using methods as previously described35 (link). Briefly, RNA sequencing libraries were prepared with TruSeq Stranded mRNA Library Prep Kit (Illumina) using 500 ng – 1 µg RNA. Quality of libraries were validated on an Agilent Bioanalyzer using DNA 1000 reagents and chips (Agilent Genomics, Waldbronn, Germany) to quantify library sizes and confirm the absence of primer dimers. Libraries were quantified using a KAPA Universal Library Quantification kit (Roche Diagnostics Limited) on a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) and library concentrations were adjusted for library size. Pooled libraries of 12–15 pM concentrations were sequenced on a MiSeq System (Illumina) using a MiSeq Reagent Kit v3 (Illumina) with 2 × 75 cycles.
Dna 1000 reagents and chips
Manufactured by Agilent Technologies
Sourced in Germany
The DNA 1000 reagents and chips from Agilent Technologies are designed for the analysis of DNA samples. They provide a reliable and accurate method for the quantification and sizing of DNA fragments ranging from 25 to 1,000 base pairs. The reagents and chips are compatible with Agilent's Bioanalyzer instruments, enabling efficient DNA analysis within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Truseq stranded mrna library prep kit, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Miseq system, by Illumina (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Universal kapa library quantification kit, by Roche (1 mentions)
Ampliseq for cancer hotspot panel v2, by Illumina (1 mentions)
Agilent 2100 bioanalyzer device, by Agilent Technologies (1 mentions)
2 protocols using dna 1000 reagents and chips
1
Whole transcriptome RNA-seq library preparation
2
Targeted Cancer Mutation Profiling using NGS
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Following the manufacturer instructions, the libraries were prepared using AmpliSeq™ for Illumina Cancer Hotspot Panel v2 (Illumina, Inc., US, Cat. No.: 20019161) which is a targeted next generation sequencing assay detecting actionable mutations across the hotspot regions of 50 genes. Quality of the libraries was checked out by Agilent 2100 Bioanalyzer device utilizing the DNA 1000 reagents and Chips (Agilent Technologies, Santa Clara, California, Cat. Code: 5067-1504). The expected PCR product is 186-277 bp, which indicates successful library amplification. Patients' libraries together with PhiX control library were normalized and equal volumes were pooled to form the terminal sequencing library. The AmpliSeq™ for Illumina Cancer Hotspot Panel achieves detection limits of 5% variant allele frequency across 207 amplicons with > 95% of bases covered at ≥ 500× [14] . Sequencing was done using Cancer Hotspot Panel v2 Nano kit on MiSeqDx device (llumina) with a 2 × 150 bp read length and total time of ~17 h which involve cluster generation, sequencing, and base calling on the MiSeqDx system.
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