GSM5202203: Ctrl1; Homo sapiens; RNA-Seq
The bone marrow samples from SF3B1 mutated MDS were harvested by bone marrow aspiration. The bone marrow samples from healthy donors were harvested from femoral head after patients underwent hip surgery. Following the sample, the mononuclear cells were isolated on a Ficoll gradient. Once isolated, mononuclear bone marrow cells were counted and resuspended in an adequate solution (IMDM, SVF 40%, DMSO 15%) and frozen in liquid nitrogen at a concentration of 20 to 30 million cells per mL. Bone marrow mononuclear cells were thawed, and dead cells were eliminated by immunomagnetic negative sorting (MACS MicroBead technology from Miltenyi Biotec ™). A second CD34 positive immunomagnetic sorting was performed to isolate stem and progenitor cells. Cells were then washed with PBS containing 0.04% of BSA. Cell concentration and viability were determined microscopically with a Malassez counting chamber cell after staining with trypan blue. Libraries were prepared with the chromium system of 10x genomics, with the Chromium Single Cell 3 'V2 kit according to the manufacturer's protocol (www.10xgenomics.com). The four samples (Ctrl1, Ctrl3, MDS2 and MDS4) were processed on the same chromium chip. The number of cells targeted per sample was 5000 cells. The libraries were sequenced by the Integragen company on an Illumina HiSeq4000 sequencer with a target depth of 50,000 reads per cell.