Single-cell Sequencing Analysis of Head and Neck Squamous Cell Carcinoma

To better understand tumor microenvironment and tumor heterogeneity in head and neck squamous cell carcinoma, we profiled primary tumors from two treatment naive patients by single-cell sequencing. Primary tumors from two treatment naive patients, and fresh HNSCC tissues were dissociated with the Gentle MACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. Next, single cells were counted using automated cell counter and adjusted to the concentration of 1000 cells/μL and we retained single-cell transcriptomes for 13,903 cells after initial quality controls. Cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Cells were lysed, and the RNAs were reverse-transcribed into cDNAs. Libraries were generated from the cDNAs with Chromium Next GEM Single Cell 3’ Reagent Kits v3.1. Finally, the cDNA libraries were sequenced using the nova 6000 Sequencing System (Illumina). The SRA Study accession is SRP293588 and the BioProject accession is PRJNA679702.