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Analysis of single-cell RNA-seq data from human PBMCs and from in vitro cultures of human cord blood CD34+ progenitors encompassing different DC types

ABSTRACT: For both PBMC and cells from the in vitro cultures, RNA purification and library generation was performed using the Chromium Single Cell Controller apparatus and associated protocols (10X Genomics). Libraries were sequenced by 75-bp single-end reading on a NextSeq500 sequencer (Illumina). Reads were aligned on the GRCh38 human genome assembly. Data analysis was performed using the R software package Seurat (https://github.com/satijalab/seurat)

ORGANISM(S): Homo sapiens

PROVIDER: GSE115405 | GEO | 2018/08/14

REPOSITORIES: GEO

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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.

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Analysis of single-cell RNA-seq data from human PBMCs and from in vitro cultures of human cord blood CD34+ progenitors encompassing different DC types

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