GSM8901046: A126899; Homo sapiens; RNA-Seq
Fresh surgical specimens were immediately transferred to the laboratory in ice-cold RPMI 1640 medium.  Tissues were manually minced and enzymatically dissociated using a cocktail of collagenase type I, DNase I, and hyaluronidase at 37°C with gentle agitation to generate single-cell suspensions.  Following enzymatic digestion, cell suspensions were filtered through a 40 μm strainer, centrifuged, and resuspended in PBS with 0.04% BSA.  Cell viability was assessed using trypan blue staining, and samples with viability >85% were processed for library preparation using the 10x Genomics Chromium Single Cell 3' protocol according to the manufacturer's instructions. Single-cell suspensions were processed using the 10x Genomics Chromium Single Cell 3ʹ v3 (or v3.1, if applicable) protocol. Cells were loaded onto the Chromium Controller to generate gel bead-in-emulsions (GEMs), enabling barcoding of individual cells. Reverse transcription, cDNA amplification, and library construction were performed according to the manufacturer's instructions. Final libraries were quantified using a Bioanalyzer (Agilent) and Qubit (Thermo Fisher), and sequenced on an Illumina NovaSeq 6000 platform with paired-end reads.