GSM8388921: P5, Baseline, scRNA-seq; Homo sapiens; RNA-Seq
The BM aspirate samples were collected during the diagnostic procedure. Bone marrow mononuclear cells (BMMCs) were isolated using Ficoll Paque Plus (Cytiva) and washed three times with Hanks Balanced Salt Solution (HBSS, Solarbio). Following centrifugation at 1,000 rpm for 5 min, the supernatant was discarded, and the sediment was resuspended in 1 ml phosphate-buffered saline (PBS, Solarbio). To eliminate red blood cells, 1-2 ml red blood cell lysis buffer (BD) was added at 4°C for 10 min. After centrifugation at 500 g for 5 min, the cells were suspended in PBS. The cell viability was assessed using trypan blue (Sigma) and examined under a microscope. Utilizing the 10x Genomics Chromium Single Cell 3′ v3 Library Kit and Chromium instrument, approximately 17,500 cells were partitioned into nanoliter droplets to achieve single-cell resolution for a maximum of 10,000 individual cells per sample. The resulting cDNA was tagged with a common 16 nt cell barcode and 10 nt Unique Molecular Identifier during the RT reaction. Full-length cDNA from poly-A mRNA transcripts was enzymatically fragmented and size selected to optimize the cDNA amplicon size (approximately 400 bp) for library construction (10x Genomics). The concentration of the 10x single-cell library was accurately determined through qPCR (Kapa Biosystems) to produce cluster counts appropriate for the HiSeq4000 or NovaSeq6000 platform (Illumina). In all, 26 × 98 bp (3′ v3 libraries) sequence data were generated targeting between 25 and 50K read pairs/cell, which provided digital gene expression profiles for each individual cell.