Characterization of human erythropoiesis using ex vivo differentiation of CD34 HSC (RNA-seq)

Genome Wides Association Studies (GWAS) have identified tens of thousands of associations between human genetic variation and common disease. Despite the abundance of GWAS associations, functional identification and characterization of causative variants and effector genes remains a challenging prospect. Human erythropoiesis provides a highly tractable model system for the development of tools for GWAS analysis. We have developed a three-phase protocol for differentiation of peripheral CD34+ Haematopoietic Stem Cells (HSC) into mature enucleating erythrocytes. This protocol has been characterized for it's the changes observed morphologically, immunologically (by FACS) and epigenetically (by ATAC-seq, ChIP-seq, and RNA-seq). We have used this protocol to study the effects of common GWAS variants affecting red blood cell traits, and more severe mutations causing Type-1 Congenital Dyserythropoietic Anemia (CDA-I). Overall design: To characterise the changes in gene expression during erythropoiesis, PolyA+ RNA-seq was carried out at days 7, 10, and 13 of HSC differentiation. CD34+ HSC were differentiated from three separate donors on multiple occasions with single replicates (rep1 and rep2) performed for each differentiation.