GSM8910959: STA_TCR, epilepsi, #1; Homo sapiens; RNA-Seq
The excised vascular tissue was immediately washed three times in Dulbecco's Phosphate-Buffered Saline (DPBS) to remove blood and debris, and then transported on ice for processing within 2 hours. If immediate processing was not possible, samples were stored in 4°C DPBS (no longer than 6 hours). Under a sterile laminar flow hood, the tissue was cut into approximately 0.5 mm³ pieces and incubated in enzyme digestion solution (collagenase II 1 mg/ml, DNase 50 µg/ml, hyaluronidase 0.1 mg/ml) at 37°C for 30-60 minutes, with gentle pipetting every 10 minutes to promote dissociation. The digested suspension was filtered through a 70 µm mesh filter to remove undigested fragments and collected into 15 ml centrifuge tubes. Red blood cell contamination was removed using red blood cell lysis buffer, and cell viability was assessed using trypan blue staining. Live cells (trypan blue-negative) were sorted using flow cytometry (FACS) to ensure high-quality sequencing. Cell concentration was determined using a hemocytometer to ensure each sample contained 10⁵-10⁶ cells/ml. Single-cell suspensions were processed using the 10X Genomics Chromium System (10x Genomics, Pleasanton, CA, USA) to create 3' gene expression libraries with the Chromium Single Cell 3′ Reagent Kit v3. Briefly, single-cell suspensions, reagents, gel beads, and partitioning oil were loaded onto a 10X Chromium Chip B. Within the chip, each cell was encapsulated in an oil droplet containing gel beads and reagents, forming a gel bead in emulsion (GEM). Each GEM serves as an independent reaction chamber where cell lysis and reverse transcription of poly-adenylated mRNA into barcoded full-length cDNA occur, with primers incorporating a sequencing primer, a cell-specific barcode, unique molecular identifiers (UMIs), and a poly(dT) tail. All cDNA molecules were then pooled, and standard library construction steps followed, including amplification, fragmentation, end repair, adapter ligation, and sample index PCR. The libraries were sequenced on an Illumina NovaSeq 6000 platform using paired-end sequencing (PE150).