GSM8966567: Sample 6063 leukemic blasts; Homo sapiens; RNA-Seq
Patient samples were thawed and washed with PBS supplemented with 5% FBS (Corning) supplemented with DNase I (Sigma) at 50mg/mL. Cells were incubated with a fixable viability stain 510 (FVS510, BD Biosciences) for 15 min at room temperature, washed, and then stained with antibodies against human CD45 (BD Horizon BV786), CD33 (BD Horizon BV421), CD3 (BD Horizon APCR700), and CD19 (BD Horizon BB515) for 20 min at room temperature. Cells were then washed and resuspended in PBS supplemented with 2% FBS and flow sorted to isolate viable (FVS510−) CD3−/CD19−/CD45+ / CD33+ cells using a BD FACSAria III cell sorter. Following flow sorting, primary samples were washed in PBS and then extraction was performed using the Qiagen AllPrep DNA/RNA kit. The purity and concentration of the extracted DNA and RNA were assessed using a Nanodrop before downstream applications. RNA was sent to a commercial provider, Tempus, for library preparation and sequencing on an Illumina HiSeq. RNA libraries were prepared from PolyA selected mRNA species