NextSeq 500 paired end sequencing; Single-cell RNAseq of human bone marrow cells enriched for stromal and endothelial markers
Single-cell RNAseq of human bone marrow cells enriched for stromal and endothelial markers
F80_CD144_p
Excess bone marrow from patients undergoing hip-replacement surgery was used. Samples were stored at 4°C over night and bone marrow mononuclear cells were isolated as described in Kanczler et al. 2019 (PMID: 30729460). In addition, whole bone marrow was diluted 1:1 with a-MEM (Gibco) digested with collagenase IV (20U/mL, Thermo Fisher, 17104019) for 30min at 37C under continuous rotation. Where indicated, magnetic cell sorting (MACS) was conducted to enrich or deplete cells. Up to a total of 1x10^8 BM-MNCs were used for each separation. Cells expressing CD45 (Miltenyi Biotec, 130-045-801), CD146 (Miltenyi Biotec, 130-093-596), CD144 (Miltenyi Biotec, 130-097-857), CD106 (Miltenyi Biotec, 130-104-123 and 130-048-102) were isolated using LS columns (Miltenyi Biotec, 130-042-401) according to manufacturer's instructions. Single-cell transcriptomes were isolated using the DropSeq protocol described in Macoscko et al. 2015 (PMID:26000488). Protocols for NGS library preparation described in Macosko et al. 2015 (PMID:26000488) were closely followed and pre-amplification was conducted using 4+12 PCR cycles (95°C 3 minutes - 4 cycles of: 98°C 20s; 65°C 45s; 72°C 3min - 12 cycles of: 98°C 20s; 67°C 20s; 72°C 3min - 72°C 5min; 4°C hold).