GSM7901366: CML 339 FLU + NIL; Homo sapiens; RNA-Seq
For isolation of stem cells (CD34+CD38-), CD34+ cell samples were stained with anti-human (hu) CD34-APC, CD38-V450 or CD38-Pe-Cy7 antibodies (BD Biosciences), before being subjected to fluorescence activated cell sorting (FACS) to obtain the stem cell populations using a FACSAriaTM Fusion cell sorter (BD Biosciences). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. Libraries were quantified using the Agilent Bioanalyzer High Sensitivity DNA Kit.