title: GSM8630045: Pool1_Mouse2_Primary_PDX; Homo sapiens; RNA-Seq
library_construction_protocol: Mice were euthanized upon reaching pre-determined clinical endpoints such as such as significant body weight loss, seizures, and a persistent hunched posture with diminished activity. The collected mice brains were imaged with a fluorescent stereomicroscope with 0.63 objective magnification (Olympus MVX10, Leica M205), which covers both the entire brain and cell morphology at single-cell resolution. Under the microscope, the brains were observed from the ipsilateral hemisphere for tumor-peripheral invasion to the contralateral hemisphere for distal invasion. For distal invasion, collected brains were physically separated into the hemisphere with the primary tumor (injection side) and the other contained invading tumor cells excluding the midline border region. For local blood vessel-associated invasion, tube-structured GFP+ cell clusters were dissected under a stereomicroscope without introducing any primary bulk tumor mass. Then, each location were mechanically and enzymatically dissociated using a papain-based neural tissue dissociation kit (Miltenyi Biotec) supplemented with 0.1% Type I collagenase (Thermo Fisher Scientific)/PBS. The dissociated cells were first stained with calcein Blue AM (BD Biosciences) and Zombie NIR (BioLegend) for 25 min at 4C, and with anti-mouse CD16/32 (BD Biosciences) for 5 min. After washing cells with ice-cold 1% BSA/PBS, the cells were then stained with PerCP anti-mouse CD45 (clone 30-F11, BD Biosciences) for 30 min at 4C. Sorting was performed with Becton Dickinson Influx cytometer (Becton Dickinson). Side scatter (SSC) width versus forward scatter (FSC) area and Trigger Pulse Width versus FSC criteria were used to discriminate doublets and gate-only singleton cells. Viable single cells were identified as calcein blue AM positive and Zombine NIR negative to low cells. We sorted viable CD45 negative/GFP positive cells from blood-vessel associated invasion samples into 96-well plates containing 10uL of TCL buffer (QIAGEN) with 1% beta-mercaptoethanol (Sigma Aldrich). Plates were frozen immediately after sorting and stored at -80 degree prior to whole transcriptome amplification, library preparation, and sequencing. For multiplexed samples, CD45 negative/GFP positive cells were sorted into 15mL tumors for subsequent 10X scRNA-seq processing. The spheroid models in vitro were processed as a pool using TotalSeq-B/Hashtag anti-bodies (BioLegend), described previously19. Each of the dissociated cells was first stained with HumanTruStain FcX (BioLegend) in Cell Staining Buffer (BioLegend) for 10 min at 4C, and then stained with barcoded TotalSeq-B anti-bodies targeting human CD298 and b2 microglobulin in a separated tube for 30 min at 4C. We generated a single cell suspension of the pool in 0.04% PBS-BSA and loaded approximately 9,900 single cells to the Chromium Controller with a targeted recovery of 6,000 cells. Single cells, reagents and single gel beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized droplets and subjected to reverse transcription. Droplets were broken and the barcoded cDNAs were purified with DynaBeads and amplified by 12 cycles of PCR (98C for 3 min; [98C for 15 s, 63C for 20 s, 72C for 1 min] x 12; 72C for 1 min). Gene expression cDNA and TotalSeq antibody derived barcode cDNA were size-selected and separated with SPRIselect Reagent (Beckman Coulter), and then fragmented, end-repaired, ligated with index adaptors. Quality control of the resulting libraries was performed with the Bio-analyzer High Sensitivity DNA Analysis (Agilent). The constructed gene expression library and barcode library were combined at 4:1 ratio and sequenced with paired-end, 28 and 55-base reads, using a NextSeq 2000 sequencer (Illumina).