GSM9024944: s004; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated using standard density gradient centrifugation. Following isolation, cells were filtered according to the 10x Genomics sample preparation protocol to remove debris and aggregates. Only highly viable cells were selected for downstream single-cell RNA sequencing. The sequencing protocol adhered to the recommended guidelines of the 3' V3 chemistry kit by 10X Genomics. Initially, single-cell Gel Beads-in-Emulsion (GEMs) were generated by combining 350K – 400K PBMCs, barcoded Single Cell 3' v3.1 beads, and partitioning oil. These GEMs underwent processing via a 10X Chromium Controller machine (10X Genomics). Subsequent steps encompassed gel bead elimination, cell lysis, and reverse transcription in a C1000 Touch Thermal Cycler (BioRad) to yield barcoded cDNA. To extract oil residues, the GEM mixture underwent purification using Dynabeads, followed by cDNA amplification. Post-amplification cDNA was purified using the SPRIselect reagent. Evaluation of cDNA quality and quantity occurred via a 2100 Bioanalyzer Instrument (Agilent Technologies). Further stages entailed fragmentation, end repair, A-tailing, and adaptor ligation in accordance with the manufacturer's protocol. Library quality was verified using the Bioanalyzer. Final libraries were sequenced on the Illumina NovaSeq 6000 platform using paired-end reads to achieve high-throughput transcriptomic profiling at single-cell resolution.