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C8ca7ee1-57d2-4692-94da-2126b20ffc70 - samples

ABSTRACT: 294 formalin-fixed paraffin-embedded (FFPE) tissue samples were sent to the UNC Lineberger Comprehensive Cancer Center Translational Genomics Lab (TGL) for RNA isolation using the Maxwell 16 MDx Instrument (Promega AS3000) and the Maxwell 16 LEV RNA FFPE Kit (Promega AS1260) following the manufacturerÃ¢Â€Â™s protocol (Promega 9FB167). 279 total RNA sequencing libraries were prepared at TGL using a Bravo Automated Liquid-Handling Platform (Agilent G5562A) and the TruSeq Stranded Total RNA Library Prep Gold Kit (Illumina 20020599) following the manufacturerÃ¢Â€Â™s protocol (Illumina 1000000040499). RNAseq library quality and quantity were measured using a TapeStation 4200 (Agilent G2991AA) and Qubit 3.0 fluorometer (Life Technologies Q33216), pooled at equal molar ratios, and denatured following the manufacturerÃ¢Â€Â™s protocol (Illumina 1000000106351). 271 total RNA sequencing libraries were sequenced at TGL on NovaSeq 6000 (Illumina 20012850) S4 flow cells (Illumina 20028313) following the manufacturerÃ¢Â€Â™s protocol (Illumina 1000000019358) using a 2x50 bp paired-end configuration and pool sizes of 91 libraries to target a read depth of 110 million clusters per library on average.

PROVIDER: EGAD00001010201 | EGA |

REPOSITORIES: EGA

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Project description:Purpose: To investigate the diet- or strain-dependence of liver transcriptome in eight Diversity Outbred (DO) founder strains [A/J, C57BL/6J (B6), 129S1/SvImJ (129), NOD/ShiLtJ (NOD), NZO/HILtJ (NZO), and three wild-derived strains CAST/EiJ (CAST), PWK/PhJ (PWK), and WSB/EiJ (WSB)] Methods: Total RNA from eight DO founder strains was extracted from snap frozen liver using Maxwell® 16 LEV simplyRNA Tissue Kit (Promega) according to the manufacturer’s protocol. The quality and amount of liver RNA was evaluated using a qubit RNA assay (Invitrogen R11490) and run on an Biorad Bioanalyzer Chip to verify RNA integrity. RNASeq library was prepared by using Illumina TruSeq RNA library construction protocol. RNA samples from 24 mice-fed AIN-93 M diet and 24 mice-fed HFCA diet were submitted to the David H. Murdock Research Institute (DHMRI) for sequencing. The RNA-seq libraries were constructed from 1 µg total RNA after poly-A library preparation. The pooled libraries were sequenced on two lanes of the Illumina HiSeq2500 Instrument. Results: Our liver transcriptomic analysis of eight mouse strains shows that diet and genetic background have a strong effect on the liver transcriptome, which may be related to differences in cardo-metabolic traits. We calculated a gene module consisting of highly correlated transcripts that enriched the biological pathway and provided a searchable database of liver transcript profiles. To further investigate the association of hepatic gene modules with cardio-metabolic traits, Fmo3, a key enzyme in the production of trimethylamine N-oxide (TMAO), and Nox4, which showed strong association with plasma TMAO and liver triglyceride, were identified as the highest expressed in the laboratory-like inbred strains (B6, 129 and NZO) and the lowest expressed transcripts in the CAST strain. We found differences in the production of TMAO and liver triglyceride, which contributes to metabolic syndrome, depending on genetic backgrounds. Conclusions: Our study provide a valuable data resource to the research community and show that liver transcriptomic analysis identified diet- or strain-specific pathways to pathogenesis of metabolic syndrome.

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C8ca7ee1-57d2-4692-94da-2126b20ffc70 - samples

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