GSM9371466: HCC PBMC patient B-05 timepoint C1D1 scRNA-seq; Homo sapiens; RNA-Seq
Blood samples were processed using Ficoll (Cytiva); after centrifugation, the PBMC layer was isolated and cryopreserved in cell media with human serum and DMSO. Previously frozen PBMCs were thawed using media containing RPMI, heat-inactivated sterile filtered fetal bovine serum, penicillin-streptomycin, non-essential amino acids, sodium pyruvate, and L-glutamine. 1.1x105 cells from each sample were combined into staining buffer (1 mmol/L EDTA and 2% heat-inactivated FBS in calcium/magnesium-free Dulbecco PBS; Thermo Fisher Scientific) and stained with one pooled cocktail containing a custom panel of 197 Total-Seq C antibody–oligonucleotide conjugates (BioLegend) as per standard protocols following pre-incubation with TruStain FcX (Fc Receptor Blocking Solution, Biolegend). Droplet-based single cell RNA sequencing (scRNAseq) was performed using the 10x Genomics Chromium Single Cell 5ʹ Reagent Kits v2, according to manufacturer instructions. 10X Genomics 5′ v2 Library Construction kits for feature barcoding (protein antibodies) or CITE-seq were used to generate libraries per manufacturer instructions. All sequencing was performed on an Illumina NovaSeq S4 sequencer with paired end 200 base pair read length and 25,000 reads per droplet for gene expression libraries and 5,000 reads per cell for feature barcode libraries.