GSM8724605: PTEN MUT 3; Homo sapiens; RNA-Seq
Human samples were obtained from the Nervous System Tumor Bank at Northwestern University. In brief, patients diagnosed with GBM underwent surgery for tumor resection following standard clinical practices. After the tumor was removed, an adequate amount of tissue was sent to pathology for clinical diagnosis. During the surgery, a maximum of 60 mL of peripheral blood was collected while the patient was under anesthesia. Blood was drawn by the anesthesiologist from an arterial line, central venous line, or a peripheral IV, all of which were part of routine anesthesia care.  The peripheral blood was then centrifuged using a Ficoll gradient to separate the peripheral blood leukocytes and plasma, which were collected and frozen separately. Before analysis, the peripheral blood mononuclear cells (PBMCs) were retrieved from liquid nitrogen and thawed in a 37°C water bath. The cells were washed with phosphate-buffered saline (PBS) and subjected to the EasySep™ Dead Cell Removal (Annexin V) Kit (STEMCELL, #7899), following the manufacturer's protocol. The viable cells were then sent to the NUSeq Core at Northwestern University for scRNA-seq analysis. The scRNA-seq library was prepared by Northwestern University NUseq facility core with NIH Grant (1S10OD025120). To ensure the quality of library, the number and viability of human GBM patients' PBMCs were stained with AO/PI Staining Solution and analyzed by Nexcelom Cellometer Auto2000 cell viability analyzer. PBMCs were loaded into the Chromium Controller (10X Genomics, PN-120223) on a Chromium Next GEM Chip K (10X Genomics, PN-1000127), and processed to generate single cell gel beads in the emulsion (GEM) according to the manufacturer's protocol. The cDNA and library were generated using the Chromium Next GEM Single Cell 5' Reagent Kits v2 (10X Genomics, PN-1000283) according to the manufacturer's manual.