Single Cell RNA-Seq of Neoadjuvant Pembrolizumab Clinical Trial HNSCC Patient Tumors
Fresh tumors were mechanically and enzymatically disaggregated in dissociation buffer containing collagenase type IV and DNase I. Cells were strained through a 40um filter, incubated with viability dye Zombie Green, Fc receptors blocked, and then stained for human CD45 and human CD3. Samples were washed twice and then sorted to isolate viable (Zombie-negative) CD45+ CD3+ lymphocytes. Cell density was adjusted to analyze approximately 10,000 cells per sample. Single-cell gene expression (scRNA-seq) was performed using Chromium Single Cell 5-prime Library and Gel Bead Kit (10x Genomics). Gel Bead-in-Emulsion reverse transcription (GEM-RT) reaction, clean-up and PCR amplification was performed to obtain cDNA for RNA-seq library generation. TCR V(D)J targeted enrichment library preparation (Chromium Single Cell V(D)J Enrichment Kit, Human T cell) was then performed. Single cell sequencing libraries were generated using 5-prime GEXv1 plus V(D)J enrichment according to manufacturer protocol. Libraries were uniquely indexed for multiplexed sequencing.