Illumina HiSeq 2500 paired end sequencing; GSM3145849: Ghrelin_CTR_Liver_male_1; Mus musculus; RNA-Seq

Ghrelin_CTR_Liver_male_1

Gut microbiota and the circadian clock are both key regulators of the metabolic processes. Although recent evidence points to the impact of the circadian clock on microbiota, gut microbiota effect on diurnal host gene expression remains elusive. A transcriptome analysis of germ-free mice reveals subtle changes in circadian clock gene expression. However, a lack of microbiome leads to liver feminization and alters the expression of male-specific genes involved in lipid metabolism and xenobiotic detoxification associated with sustained activation of the Growth Hormone pathway. These results emphasize the mutual interaction of gut microbiota and its host even on unexpected functions. Overall design: Total RNA-Seq of Germ-free (GF) male mice liver injected with ghrelin.

Total RNA-Seq of Germ-free (GF) male mice liver injected with ghrelin.

Submitted on 02-SEP-2018

Liver RNAs were extracted as previously described (Gachon et al, 2006) with classical methods using guanidium  thiocyanate lysis buffer. The RNA were quantified with Ribogreen (Life Technologies) and quality assessed on a Fragment Analyzer (Advances Analytical). Sequencing libraries were prepared from 250 ng total RNA using the TruSeq Stranded Total LT Sample Prep Kit (Illumina) with the Ribo-Zero Gold depletion set. The procedure was automated on a Sciclone NGS Workstation (Perkin Elmer). The manufacturer's protocol was strictly followed, except for the PCR amplification step. The latter was run for 12 cycles with the KAPA HiFi HotStart ReadyMix (Kapa BioSystems). This optimal PCR cycle number has been evaluated using the Cycler Correction Factor method as described previously (Atger et al., PNAS, 2015). Purified libraries were quantified with Picogreen (Life Technologies) and the size pattern was controlled with the DNA High Sensitivity Reagent kit on a LabChip GX (Perkin Elmer). Libraries were then pooled by 24 and each pool was clustered at a concentration of 7 pmol on 8 lanes of a v3 paired-end sequencing flow cell (Illumina). Sequencing was performed for 2 x 125 cycles on a HiSeq 2500 strictly following Illumina's recommendations.

Total RNA-Seq of Germ-free (GF) male mice liver injected with ghrelin.

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