Sequencing libraries were constructed using TruSeq DNA V2 (Illumina, San Diego, CA) sample prep kits and quantified using qPCR (Kapa Biosystems, Wilmington, MA). The mRNA was fragmented, and double-stranded cDNA was generated by random priming. The ends of the fragmented DNA were converted into phosphorylated blunt ends. An ‘A’ base was added to the 3′ ends. Illumina®-specific adaptors were ligated to the DNA fragments. Using magnetic bead technology, the ligated fragments were size-selected and then a final PCR was performed to enrich the adapter-modified DNA fragments, since only the DNA fragments with adaptors at both ends will amplify. Libraries were pooled and sequenced by the Genome Technologies core facility at The Jackson Laboratory. Samples were sequenced on Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents (Illumina), targeting 30 million read pairs per sample. Samples were split across multiple lanes when being run on the Illumina HiSeq, once the data was received the samples were concatenated to have a single file for paired-end analysis.
Truseq dna v2
Manufactured by Illumina
Sourced in United States
The TruSeq DNA V2 is a library preparation kit designed for high-quality, multiplexed sequencing of DNA samples. It provides a streamlined workflow for generating indexed DNA libraries from a variety of input DNA amounts. The kit includes reagents and protocols for DNA fragmentation, end-repair, A-tailing, adapter ligation, and PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 3000 4000 sbs kit reagents, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Adaptor, by Illumina (1 mentions)
Hisequation 2500, by Illumina (1 mentions)
Fastprep 24 homogenizer, by MP Biomedicals (1 mentions)
Masterpure yeast dna purification kit, by Illumina (1 mentions)
Truseq nano dna kit, by Illumina (1 mentions)
5 protocols using truseq dna v2
1
Illumina mRNA Sequencing Library Prep
2
RNA-Seq Library Preparation for Brain Tissue
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Total RNA was extracted from snap-frozen right brain hemispheres using Trizol (Invitrogen, Carlsbad, CA). mRNA was purified from total RNA using biotin-tagged poly dT oligonucleotides and streptavidin-coated magnetic beads and quality was assessed using an Agilent Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA).
RNA-Sequencing Assay Library Preparation Sequencing libraries were constructed using TruSeq DNA V2 (Illumina, San Diego, CA) sample prep kits and quantified using qPCR (Kapa Biosystems, Wilmington, MA). The mRNA was fragmented, and double-stranded cDNA was generated by random priming. The ends of the fragmented DNA were converted into phosphorylated blunt ends. An “A” base was added to the 3’ ends. Illumina-specific adaptors were ligated to the DNA fragments. Using magnetic bead technology, the ligated fragments were size-selected and then a final PCR was performed to enrich the adapter-modified DNA fragments since only the DNA fragments with adaptors at both ends will amplify.
RNA-Sequencing Assay Library Preparation Sequencing libraries were constructed using TruSeq DNA V2 (Illumina, San Diego, CA) sample prep kits and quantified using qPCR (Kapa Biosystems, Wilmington, MA). The mRNA was fragmented, and double-stranded cDNA was generated by random priming. The ends of the fragmented DNA were converted into phosphorylated blunt ends. An “A” base was added to the 3’ ends. Illumina-specific adaptors were ligated to the DNA fragments. Using magnetic bead technology, the ligated fragments were size-selected and then a final PCR was performed to enrich the adapter-modified DNA fragments since only the DNA fragments with adaptors at both ends will amplify.
3
NGS Library Preparation Protocol
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Sequencing libraries were constructed using TruSeq DNA V2 (Illumina, San Diego, CA) sample prep kits and quantified using qPCR (Kapa Biosystems, Wilmington, MA). The mRNA was fragmented, and double-stranded cDNA was generated by random priming. The ends of the fragmented DNA were converted into phosphorylated blunt ends. An “A” base was added to the 3’ ends. Illumina®-specific adaptors were ligated to the DNA fragments. Using magnetic bead technology, the ligated fragments were size-selected and then a final PCR was performed to enrich the adapter-modified DNA fragments since only the DNA fragments with adaptors at both ends will amplify.
4
Comprehensive Cryptococcus neoformans Isolation and Sequencing
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C. neoformans was isolated from HIV-infected individuals on location by plating CSF onto Sabourand Dextrose (SD) agar (Oxoid, Fisher Scientific), and growing at 30° for 48 hr. A representative sample of the C. neoformans population was taken by selecting a broad “sweep” of all colonies on the SD agar plate, which was stored in cryopreservative medium (80% SD broth, 20% glycerol) at −80° until further testing. This approach ensures that all genetic diversity is maintained through the process, and single colony picking only occurs at the final stage of liquid culture and DNA extraction.
Frozen stocks were plated onto SD agar and cultured for 72 hr. A single colony was inoculated into 6 ml Yeast Peptone Digest broth (Oxoid) supplemented with 0.5 M NaCl and cultured at 37° with agitation (165 rpm) for 40 hr, followed by genomic DNA extraction using the Masterpure Yeast DNA purification kit (Epicentre) modified by addition of two cycles of rapid bead beating (45 sec at 4.5 m/sec) using a FastPrep 24 homogenizer (MP Bio). Genomic DNA libraries were prepared using the TruSeq DNA v2 or TruSeq Nano DNA kit (Illumina), and WGS was performed on an Illumina HiSequation 2500 at the Medical Research Council Clinical Genomics Centre (Imperial College London) as previously described (Rhodes et al. 2014 (link)).
Frozen stocks were plated onto SD agar and cultured for 72 hr. A single colony was inoculated into 6 ml Yeast Peptone Digest broth (Oxoid) supplemented with 0.5 M NaCl and cultured at 37° with agitation (165 rpm) for 40 hr, followed by genomic DNA extraction using the Masterpure Yeast DNA purification kit (Epicentre) modified by addition of two cycles of rapid bead beating (45 sec at 4.5 m/sec) using a FastPrep 24 homogenizer (MP Bio). Genomic DNA libraries were prepared using the TruSeq DNA v2 or TruSeq Nano DNA kit (Illumina), and WGS was performed on an Illumina HiSequation 2500 at the Medical Research Council Clinical Genomics Centre (Imperial College London) as previously described (Rhodes et al. 2014 (link)).
5
Transcriptome Profiling of 5XFAD Mice
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The RNA from a total of 36 5XFAD and 36 littermate control mice was sequenced; 6 mice per sex, genotype, and timepoint. Sequencing libraries were constructed using TruSeq DNA V2 (Illumina, San Diego, CA, United States) sample prep kits and quantified using qPCR (Kapa Biosystems). The mRNA was fragmented, and double-stranded cDNA was generated by random priming. The ends of the fragmented DNA were converted into phosphorylated blunt ends. An “A” base was added to the 3′ ends. Illumina®-specific adaptors were ligated to the DNA fragments. Using magnetic bead technology, the ligated fragments were size-selected, then a final PCR was performed to enrich the adapter-modified DNA fragments since only the DNA fragments with adaptors at both ends will amplify.
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