GSM9266328: OCSCC patient 1, peripheral blood, low density CD11b+ cells; Homo sapiens; RNA-Seq
Whole peripheral blood was collected via venipuncture into EDTA vacutainers (BD) and processed immediately. Blood was diluted 1:1 in PBS and layered onto 15 mL Ficoll-Paque (Cytiva) for density gradient centrifugation (400 × g, 30 min, room temperature, no acceleration or brake). The distal fraction containing red blood cells (RBCs) and high-density neutrophils was harvested and subjected to two rounds of RBC depletion (STEMCELL) followed by one round of RBC lysis (BioLegend), according to manufacturer instructions. The intermediate buffy coat layer containing low-density mononuclear cells and neutrophils was collected and subjected to two rounds of platelet removal by dilution in PBS with 2% fetal bovine serum (FBS) and centrifugation (120 × g, 10 min, room temperature, no acceleration or brake). CD11b⁺ cells were isolated from both high-density and low-density blood fractions using positive magnetic selection. Tumor biopsies were transported from the operating room to the laboratory and processed immediately. Tissues were minced into <1 mm fragments and enzymatically and mechanically dissociated into single-cell suspensions using the Human Tumor Dissociation Kit and gentleMACS Dissociator (Miltenyi Biotec) following manufacturer protocols. CD45⁺ leukocytes were isolated from dissociated tumor suspensions using positive magnetic selection. Isolated cells were washed in PBS, filtered through 70 μm strainers, and assessed for viability and concentration using acridine orange/propidium iodide (AO/PI) staining on a Cellometer Auto 2000 (Nexcelom). Cells were adjusted to 1,000 cells/μL and loaded onto the 10x Genomics Chromium Controller with a target recovery of 8,000 cells per sample. Cells were mixed with 5' v2 or 3' v3 Next GEM Kit reagents (10X Genomics) and single cells were captured along with barcoded gelbeads. Following reverse transcription, cDNA was amplified, and gene expression libraries were constructed according to the manufacturer's recommendations. Each DNA library was loaded into a sequencing lane on a NovaSeq system (Illumina) and was sequenced with paired-end reads of 75 bp.
GEO
GSM9266328_r1