Genome architecture in definitive erythroid cells at base-pair resolution
By using a novel chromatin conformation capture (3C) method (Micro Capture-C (MCC)), which allows physical contacts to be determined at base-pair resolution. We demonstrate interactions between different classes of regulatory elements in unprecedented detail. Overall design: Definitive erythroid cells were obtained from phenylhydrazine-treated mice. Spleens were mechanically disrupted to a single cell suspension in ice-cold PBS. To isolate late-stage erythroid cells, cells from a single spleen were resuspended in 5mL of cold PBS/10% FCS and stained with 50uL PE anti-Ter119 antibody (RUO, BD Biosciences) at 4°C for 30 min in the dark. After washing with 40mL of ice-cold PBS/10% FCS, cells were resuspended in 800 µL MACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and 200 µL of anti-PE magnetic beads (Miltenyi Biotec) and incubated for 20 min at 4°C in the dark. Ter119 positive cells were isolated via auto-magnetic-activated cell sorting (MACS) and then processed for downstream applications. Purity of cells was confirmed by flow cytometry.