GSM8506496: MGG75 GCO, GFPpos (FACS), scRNA-seq; Homo sapiens; RNA-Seq
Dissociated organoids were pooled together by model and resuspended in ice cold PBS with 1% BSA for fluorescence-activated cell sorting (FACS). Each sample was co-stained for 30 minutes with CellTrace Calcein Violet, AM (Thermo Fisher Scientific) and TO-PRO-3 (Thermo Fisher Scientific) live and dead cell markers, respectively. For each glioblastoma-cortical organoid FACS experiment, an age and batch matched cortical organoid was run as a negative gating control. GFP/tdT positive and negative fractions from each model were collected into separate 1.5 mL microcentrifuge tubes, with a minimum goal of 25,000 cells per collection tube. Sorting was performed with a FACS Aria Fusion Special Order System (Becton Dickinson). The sorting was limited to under 90 minutes per sample, and the collection tubes were maintained at 4° C for the sort duration, to minimize FACS artifacts on the downstream scRNA-seq results. Sorted, viable cell populations were immediately transferred to droplet-based (10x) single-cell RNA sequencing pipelines. For the 10X experiments, GFP/tdT positive and negative pools were centrifuged at 250 RCF for 5 mins, and the resulting cell pellets were resuspended in PBS + 0.04% BSA at a target concentration of 1 million cells per mL. The scRNA-seq libraries were generated using the Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1, Chromium Next GEM Chip G, and 10x Chromium Controller (10x Genomics) according to the 10X Single Cell 3′ v3.1 protocol. The GFP/tdT positive and negative cell populations were separately profiled for each glioblastoma-cortical organoid model. We loaded approximately 10,000 single cells to the Chromium Controller with a targeted recovery of 6,000 cells per cell population. 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 (98°C for 3 min; [98°C for 15 s, 63°C for 20 s, 72°C for 1 min] x 12; 72°C for 1 min). 3′ gene expression 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 Bioanalyzer High Sensitivity DNA Analysis (Agilent). The constructed gene expression libraries were paired-end sequenced using a NextSeq 2000 sequencer (Illumina). Smart-seq2 whole transcriptome amplification, library construction, and sequencing were performed as previously published.