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. 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).

为探究西妥昔单抗（cetuximab）在头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）中的耐药机制，我们对两名接受西妥昔单抗治疗前后的头颈部鳞状细胞癌患者的活检组织开展了单细胞RNA测序分析。我们依照制造商说明书，使用Gentle MACS解离仪（Miltenyi Biotec）对新鲜头颈部鳞状细胞癌组织进行解离。随后，采用自动细胞计数仪对单细胞进行计数，并将细胞浓度调整至1000个/μL；经初步质量控制后，我们共保留了13903个细胞的单细胞转录组数据。将细胞悬液加载至10X Genomics GemCode单细胞仪器中，该仪器可生成单细胞凝胶珠包封微滴（single-cell Gel Bead-In-EMlusion, GEMs）。对细胞进行裂解后，将RNA反转录为cDNA。使用Chromium Next GEM单细胞3’转录组试剂盒v3.1（Chromium Next GEM Single Cell 3’ Reagent Kits v3.1）从cDNA中构建测序文库。最终，采用nova 6000测序系统（Illumina）对cDNA文库进行测序。