title: Single Cell RNA-Seq of Neoadjuvant Pembrolizumab Clinical Trial HNSCC Patient Tumors: Sample HN12GEX3
design: 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 Live/Dead Zombie Green, Fc receptors blocked, and then stained for human CD45 and CD3. Samples were washed twice and then sorted to isolate viable (Zombie-negative) CD45+ lymphocytes. Cell density was adjusted to analyze ~10,000 cells per sample. Single-cell gene expression (scRNA-seq) was performed using Chromium Single Cell 3' Library and Gel Bead Kit (10x Genomics v2). Gel Bead-in-Emulsion reverse transcription (GEM-RT) reaction, clean-up and PCR amplification was performed to obtain cDNA for 3' RNA-seq library generation. Single cell sequencing libraries were generated using 3'GEXv1 according to manufacturer's protocol. Libraries were uniquely indexed for multiplexed sequencing.