Expression profiling of erythroid developmental subsets isolated from mouse fetal liver.. Mus musculus

The S1 and S3 erythroid developmental subsets were isolated using flow cytometry and the cell surface markers CD71 and Ter119 as described by Pop et. al. 2010 (PMID: 20877475) Expression profiles for S1 and S3 subsets were generated using Affymetrix GeneChips. Results were used to identify genes that are differentially expressed during erythropoiesis. Overall design: Single cell suspensions were prepared by mechanically dissociating whole fetal livers obtained from E12.5 to E13.5 Balb/C mouse embryos. Cells were stained for CD71, Ter119, and a cocktail containing lineage-specific antibodies. S1 and S3 erythroid developmental subsets were identified and isolated using flow cytometric sorting as described by Pop et. al. 2010 (PMID: 20877475). S1 and S3 subsets were isolated on 3 seperate days to generate total RNA (biological replicates). 20 ng of total RNA from each biological replicate was converted to cDNA, linearly amplified and biotinylated using Ovation reagents (Nugen, San Carlos, CA). Samples were hybridized to Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA). Microarray suite 5 (MAS5) processed sample data were normalized to the average of 18SRNA (AFFX-18SRNAMur/X00686_M_at), GAPDH (AFFX-GapdhMur/M32599_3_at) and β-actin (1419734_at) expression values. These gene expression profiles were performed as part of the manuscript by Shearstone et. al. Global DNA Demethylation During Erythropoiesis In Vivo