GSM9236566: TC_control; Homo sapiens; RNA-Seq
Single-cell suspensions were prepared from 1-week tri-cultures by adding 1 mL of Accutase per well and incubating for 15–20 minutes at 37 °C to achieve complete dissociation. Following dissociation, Accutase was diluted in 5 mL of BrainPhys medium supplemented with BDNF, GDNF, CNTF, IL-34, mCSF, and 10 μM ROCK inhibitor. The resulting suspension was passed through a 30-μm strainer to remove cell clumps, then centrifuged at 300 × g for 5 minutes. The cell pellet was resuspended in 70 μL of supplemented BrainPhys medium, kept on ice, and transferred to the core facility for downstream processing. Prior to sequencing, the suspension was washed at least three times with PBS containing 0.04% BSA, and cell viability was assessed to achieve a final concentration of 700–1200 cells/μL, following 10x Genomics guidelines. Single-cell transcriptomic analysis was performed using the 10x Chromium Next GEM Single Cell 3' Gene Expression v3.1 Dual Index platform (10x Genomics, Inc.) and sequenced on a NovaSeq 6000 system (Illumina, Inc.). Cell suspensions, reverse transcription reagents, gel beads, and partitioning oil were loaded onto a Next GEM Chip G (one sample per well), and GEM generation, barcoding, and reverse transcription were carried out on the 10x Chromium Controller. cDNA synthesis and library preparation followed the manufacturer's protocol (CG000315). At each stage, cDNA and library quality were assessed using a Bioanalyzer and Qubit. Libraries were sequenced on an Illumina NovaSeq 6000 with paired-end 100 bp reads, achieving ~50,000 paired reads per cell, with 93% of reads at Q30 (corresponding to 99.9% base call accuracy). Sequencing data were processed with Cell Ranger (v7.2.0) using the GRCh38-2020-A human reference genome for alignment. The resulting feature-barcode matrices were used for downstream analysis.