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

To investigate the mechanism of cetuximab resistance in head and neck squamous cell carcinoma (HNSCC), we performed single-cell RNA sequencing analysis in biopsy from two HNSCC patients before and after cetuximab treatment. Overall design: 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).