Pluto Bioinformatics

GSE73111: Gene expression analysis of Lgr5/Tspan8 expressing cells in the adult mammary gland

Bulk RNA sequencing

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. SOURCE: Ruijie Liu (cliu@wehi.edu.au) - Ritchie lab walter and eliza hall institute of medical research