NextSeq 500 paired end sequencing; Single-cell RNAseq of human bone marrow cells enriched using RNA probes
Single-cell RNAseq of human bone marrow cells enriched using RNA probes
Excess bone marrow from patients undergoing hip-replacement surgery was used. Samples were stored at 4°C over night and bone marrow mononuclear cells and STRO1+ cells were isolated using FACS as described in Kanczler et al. 2019 (PMID: 30729460). In addition, SNA treated cells were collected from human bone marrow using a FACS Aria cytometer (Becton Dickinson, Wokingham, UK). In total, 20 million cells were sorted from each patient sample. Cells were gated for monocytes, single cells, and Cy5 fluorescence, with the top 20% of fluorescent cells collected. Positive samples were determined to be within the top half of these Cy5 fluorescent cells and negative samples were considered to be within the bottom half of the collected cells. Collected cells were subsequently used for scRNAseq. 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+14 PCR cycles (95°C 3 minutes - 4 cycles of: 98°C 20s; 65°C 45s; 72°C 3min - 14 cycles of: 98°C 20s; 67°C 20s; 72°C 3min - 72°C 5min; 4°C hold).