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3’ RNA-seq of distal colon cells from mice treated or not with DSS and whose mothers were supplemented or not during pregnancy with the prebiotics GOS and inulin

ABSTRACT: 3’ RNA-seq of distal colon cells from mice treated or not with DSS and whose mothers were supplemented or not during pregnancy with the prebiotics GOS and inulin

PROVIDER: PRJEB56045 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR10183206.fastq.gz	Fastqsanger.gz
	ERR10183207.fastq.gz	Fastqsanger.gz
	ERR10183208.fastq.gz	Fastqsanger.gz
	ERR10183209.fastq.gz	Fastqsanger.gz
	ERR10183210.fastq.gz	Fastqsanger.gz

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Project description:Objective: The purpose of this study is to describe colonic cells differential transcriptomes and cellular pathways directly modulated by exposure to prebiotics. Prebiotic oligosaccharides are widely used as animal and human in-feed additives for their beneficial effects on the probiotic gut microbiota. A number of studies have revealed a protective effect of several oligosaccharides on dysfunctional or challenged intestinal cells models through microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods. However, thus far there was limited data assessing the direct effect of such functional foods on on whole-transcriptome of unchallenged intestinal epithelial cells. Methods: Colonic mRNA profiles of confluent Caco-2 cells exposed for 24h to GOS or FOS and their respective mono- and di-saccharides mock treatments were generated for 3 biological replicates. Library pool was sequenced on the Illlumina NextSeq500 over two NextSeq500 High Output 150 cycle kits (Illumina; FC-404-2002). Sequence reads were quality checked with FastQC (version 0.11.3). Using CLC Genomics Workbench 12.03, sequences were trimmed from adaptor, paired and good quality sequence read pairs were mapped to the Homo sapiens GRCh38 reference genome (Hg38). Transcripts counts were normalised and differential gene expression analysed using the RNAseq Analysis Module of CLC Genomics Workbench 12.03. Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis were developed to investigate the statistically significant gene sets (|FC value ≥ 1.5, FDR p-adj. < 0.05) by implementing the NIPA Enrichment R script (v0.6.7.R, https://github.com/ADAC-UoN/NIPA). RNAseq results were validated by qRT–PCR assays. Results: Sequencing generated between 45 to 134 million of 75-bp paired-end sequence reads per sample. An average of 58 million sequence reads per sample were paired and mapped to human genes (genome Homo sapiens GRCh38). For GOS exposure, 89 genes were differentially expressed (fold-change ≥ 1.5 and q-value < 0.05) between GOS and mock-treated groups. Gene ontology functional analysis revealed that genes up-regulated in the presence of GOS were involved in digestion, absorption, transmembrane transport whereas genes involved in drug and xenobiotic metabolic processes were reduced. With FOS treatment, 12 genes were differentially expressed (fold-change ≥ 1.5 and q-value < 0.05) relative to the control group and functional analysis of enriched GO terms revealed up-regulated DEGs were associated with steroid metabolic process. Conclusions: Our study details the first whole-transcriptome analysis of colonic epithelial cells exposed to the direct effect of prebiotics GOS and FOS. Our RNA-seq based characterization of distinctive or shared differentially expressed genes and enriched pathways generated for GOS and FOS would support further network analyses and enable the examination of intricate biological functions within more complex environments.

Project description:Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide (FOS)/inulin utilisation genes induced during growth on inulin included one encoding a b-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, while a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis demonstrated that the b-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked PTS II transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the utilization of inulin. The R. inulinivorans B-fructofuranosidase was over-expressed in E. coli and shown to hydrolyse fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosacharides. Genes induced on starch included the major extra-cellular a-amylase and two distinct a-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles. RNA was purified from mid-exponential phase (OD650 = 0.4) cultures of R. inulinivorans grown on basal YCFA supplemented with a single substrate of either starch or inulin using the RNeasy RNA purification kit (Qiagen), and the mRNA component enriched using the MICROBExpress system (Ambion). The purified RNA (1 ug) was labelled by reverse transcription (Amersham), employing random nonamer extension incorporating either dCTP-Cy3 or dCTP-Cy5 dyes. In order to ensure reproducibility, and to obtain statistically significant results, the dye labelling was swapped for a second hybridisation. RNA purified from a separate biological replicate was labelled and hybridised twice in the same way.

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3’ RNA-seq of distal colon cells from mice treated or not with DSS and whose mothers were supplemented or not during pregnancy with the prebiotics GOS and inulin

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