|Public on Oct 24, 2018
|femoral heads/knee bone explants
|cell type: Mesenchymal stromal cells
|Cells were allowed to adhere overnight in MSC basal medium before stimulation for 24hrs with 10 nM epidermal growth factor (EGF) alone, EGF in the presence of 10 µM IWR-1 or left untreated in 2 ml MSC basal medium.
|Primary human MSCs at p4 were plated into two wells of three 6 well plates in triplicate.
|After 24hrs, media was aspirated and total cellular RNA was extracted following the manufacturer’s guidelines using a Nucleospin RNA II kit from Macherey-Nagel having pooled cells from two wells for each RNA column to increase yield.
RNA yield was quantified and checked for quality using a Nanodrop spectrophotometer before quality control and further quantification using an Agilent bioanalyser prior to RNAseq
|Illumina HiSeq 3000
|Illumina HiSeq 3000 sequencing
Reads were trimmed using Cutadapt v.1.8.3
Reads were aligned to version GRCh38 of the human genome using HISAT2 v 2.0.5
Transcriptomes were assembled and gene expression quantified using the Tuxedo pipeline (version 2.2.1). Transcriptome assembly, merging, quantification and normalisation to fragments per kilobase per million mapped reads (FPKM) were performed using Cufflinks, Cuffmerge, Cuffquant and Cuffnorm, respectively.
An analysis of variance model with experimental condition (Control, EGF, EGF+IWR-1) as factor was fit to the FPKM values in the R stats package, followed by Tukey HSD test to calculate adjusted p-values to estimate which groups differed.
Genome_build: Human genome GRCh38 GCA_000001405.15
Supplementary_files_format_and_content: Cufflinks_FPKMs_and_TukeyHSD_results.txt: tab-delimited text file containing Cuffnorm output, including FPKM values for each gene in all 9 samples, as well as Tukey HSD results in the form of p-values.
|Oct 23, 2018
|Last update date
|Oct 24, 2018
|+44 (01904) 328767
|University of York
|Epidermal growth factor activates β-catenin via integrin-linked kinase to control proliferation of mesenchymal stromal cells.