GSM9475139: Patient 19-05_ADT timepoint T04 replicate 1 scRNAseq; Homo sapiens; OTHER
Peripheral blood mononuclear cells (PBMC) were obtained from patients with biliary tract cancer as part of a Phase II trial of pembrolizumab and GM-CSF. Blood samples were processed using ficoll; 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 human serum, penicillin-streptomycin, non-essential amino acids, sodium pyruvate, and L-glutamine. Samples were then incubated for DNAse I before washing and counting. 1 x10>6 cells from 16 unique samples were combined and stained with one pooled cocktail containing 99 AbSeq antibody-oligonucleotide conjugates (BD Biosciences) per standard protocols (Olvera JG et al., 2018), following pre-incubation with TruStain FcX (Fc Receptor Blocking Solution). Samples from different individuals and timepoints were randomly mixed across experiments to minimize batch and confounding effects. Droplet-based single cell RNA sequencing (scRNAseq) was performed using the 10x Genomics Chromium Single Cell 3ʹ Reagent Kits v3, according to manufacturer instructions. For preparation of ADT libraries, we followed the standard protocol available from Olvera JG et al., 2018 following pre-incubation with TruStain FcX (Fc Receptor Blocking Solution, Biolegend). Briefly, cell suspensions were combined with master mix and loaded into the chip with gel beads to generate the gel bead-in-emulsion (GEM), which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. Reverse transcription was performed on the poly-A RNA within each GEM. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads and amplified by PCR. To prepare libraries from cDNA, SPRIselect beads were used to isolate the correct size fragments. Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added during library construction.