Transcriptomics,Genomics

Dataset Information

141

Gene expression analysis of Lgr5/Tspan8 expressing cells in the adult mammary gland


ABSTRACT: Purpose: The aim of this study is to identify genes that are differentially expressed in the different basal cell populations defined by Lgr5 and Tspan8 (T8) expression from adult mouse mammary glands. Methods: Total RNA was extracted from sorted basal populations defined by Lgr5 and Tspan8 expression from the mammary glands of 10-week-old Lgr5-GFP-IRES-creERT2 female mice. Total RNA (50 ng) for each of the two biological replicates was used to generate libraries for whole-transcriptome analysis following Illumina's TruSeq RNA v2 sample preparation protocol. Libraries were sequenced on an Illumina HiSeq 2000 at the Australian Genome Research Facility (AGRF), Melbourne. At least 20 million 100 bp single-end reads were obtained for each sample. Reads were aligned to the mouse genome mm10 using Rsubread version 1.8.0. The number of reads overlapping each Entrez gene was counted using RefSeq gene annotation and featureCounts. Filtering and normalization used the edgeR package. Genes were filtered as unexpressed if their count per million (CPM) was less than 0.5 in fewer than 2 samples. Compositional differences between libraries were normalized using the trimmed mean of M-values (TMM) method. Differential expression was analyzed using the limma package. Counts were transformed to log2-CPM values with associated precision weights using voom. Differential expression was assessed using moderated t-statistics. Genes were considered to be differentially expressed if they achieved a false discovery rate of 5% and a fold-change in excess of 2. Overall design: Total RNA was extracted and purified from each sample (Lgr5-T8hi, Lgr5+T8-, Lgr5-T8-, Lgr5+T8hi ) and their transcriptomes analysed by RNA-Seq.

INSTRUMENT(S): Illumina HiSeq 2000 (Mus musculus)

SUBMITTER: Ruijie Liu  

PROVIDER: GSE73111 | GEO | 2017-01-31

SECONDARY ACCESSION(S): PRJNA296026

REPOSITORIES: GEO

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Publications


Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5+ basal epithelial cells, we identify a new marker, Tetraspanin8 (Tspan8). Fractionation based on Tspan8 and Lgr5 expression uncovered three distinct mammary stem cell (MaSC) subsets in the adult mammary gland. These exist in a largely quiescent state but differ in their reconstituting ability, spatial localizati  ...[more]

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