Illumina HiSeq 2500 paired end sequencing; Promoter capture Hi-C of CD34+ haematopoietic stem cells
Promoter capture Hi-C of CD34+ haematopoietic stem cells
Bone marrow was collected from patients undergoing total hip replacement (in a consented study approved by the London-Westminster Research Ethics Committee - IRAS:220344). Samples were treated with granulocyte colony-stimulation factor (G-CSF) to mobilise CD34+ cells from the bone marrow. Cells were diluted with 4 volumes of PBS with 2mM EDTA and centrifuged at 300g for 10 mins at room temperature to pellet them. The pellet was washed twice with PBS-EDTA, finally resuspended in 50ml PBS and transferred to 500 ml DMEM supplemented with 10% FBS. Cells were fixed by the addition of 26ml of 37% formaldehyde to a final concentration of 2% and fixed for 10 mins. Fixation was quenched with glycine at a 0.125M final concentration. Capture Hi-C was performed following the protocol in Mifsud et al. 2015, with the minor modification of in situ ligation instead of in solution ligation. Promoter interactions were enriched using the linked biotynilated RNA bait library. Following the Hi-C library enrichment for promoter interactions, a post-capture PCR amplification was carried out using PE PCR 1.0 and PE PCR 2.0 primers with 4 to 6 PCR amplification cycles. Using Illumina TruSeq library preparation kit.