Characterization of human erythropoiesis using ex vivo differentiation of CD34 HSC (NG Capture-C)

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 identify regulatory interactions of variants associated with red blood cell traits we performed high resolution chromatin conformation capture (3C) using NG CaptureC (Davies 2016). NG CaptureC was performed in triplicate for Day 10 differentiating erythroid (Ery) cells from three donors, human Embryonic Stem Cells (H1 hESC; WiCell), and Human Umbilical Vein Endothelial Cells (HUVEC; Lonza/Gibco/PromoCell). Capture was performed with biotinylated oligonucleotides targeting sequences adjacent to DpnII sites in two separate designs. Probes were divided into pools targeting SNPs more than (enhancer) or less than (promoter) 5 kb from known transcription start sites.