GSM8894711: Subcutaneous tumor 3, control group; Mus musculus; RNA-Seq
The experimental workflow begins with cell quality control, where the cell suspension is adjusted to an optimal concentration of 700–1,200 cells/μL and rigorously assessed for viability (>80%), minimal debris (<10%), and absence of clumping or contamination. For 10X barcoding and cDNA synthesis, cells are combined with oligonucleotide-loaded gel beads (containing 16-bp cell barcodes, 12-bp UMIs, and poly-dT primers) and encapsulated into GEMs (Gel Beads-in-Emulsion) via a microfluidic system. Within each GEM, cells are lysed, mRNA is reverse-transcribed into barcoded cDNA, and droplets are subsequently broken to pool the cDNA for amplification. The amplified cDNA is enzymatically fragmented into 200–300 bp segments, ligated with Illumina adapters (P5/P7), and size-selected to construct the sequencing library. Finally, paired-end sequencing (2×150 bp) is performed on Illumina platforms (e.g., NovaSeq 6000) targeting a depth of 25,000 reads per cell, followed by data preprocessing using Cell Ranger for demultiplexing, genome alignment, and generation of a gene-cell expression matrix. This integrated workflow ensures high sensitivity (UMI-based quantification), scalability (up to 80,000 cells per run), and compatibility with multiomic or spatial transcriptomics extensions.