Single-cell sequencing of peripheral blood and tumor-infiltrating immune cells in renal clear cell carcinoma [5' RNA expression sequencing]. Single-cell sequencing of peripheral blood and tumor-infiltrating immune cells in renal clear cell carcinoma [5' RNA expression sequencing]

We generated a comprehensive immune profile using single-cell RNA-seq data from 25,000 immune cells from three renal cell carcinoma along with matched peripheral blood. In addition, we performed VDJ sequencing on the isolated single T cells. In order to investigate heterogeneity and dynamics of cancer-associated immune cells, we performed single-cell sequencing on peripheral and tumor-infiltrating immune cells in three renal clear cell carcinoma patients. We chose renal clear cell carcinoma tumors based on the responsive of these tumors to immune checkpoint blockade in the context of low mutational loads, which implies a strong influence from the tumor microenvironment. Using the 10x Genomic 5 expression platform a total of 25,672 immune cells were isolated and passed filtering for quality control, with 13,433 cells from peripheral blood and 12,239 tumor-infiltrating cells. In addition, we used the Chromium Single Cell V(D)J kit to enrich for T cell receptor sequences for nearly 10,000 T cells in with accompanying expression information, allowing for the investigation of clonality and transcriptional phenotypic diversity. Overall design: 5' single-cell RNA sequencing performed on peripheral blood and tumor-infiltrating immune cells Single-cell RNA sequencing was performed on immune cells from three patients using the 10x genomics 5-prime expression profiling kits and TCR seauences for T cells.

本研究基于3例肾细胞癌患者及其自体匹配外周血的25000个免疫细胞的单细胞RNA测序（single-cell RNA-seq）数据，构建了全面的免疫细胞图谱。此外，本研究对分离得到的单个T细胞开展了VDJ测序。
为探究肿瘤相关免疫细胞的异质性与动态变化特征，本研究对3例肾透明细胞癌患者的外周血及肿瘤浸润免疫细胞开展了单细胞测序。本研究选取的肾透明细胞癌样本，在突变负荷较低的情况下对免疫检查点阻断治疗具有响应性，这提示肿瘤微环境对其免疫表型具有较强的调控作用。
本研究采用10x Genomics 5'基因表达平台，共分离得到25672个免疫细胞，且全部通过质量控制过滤；其中外周血来源细胞13433个，肿瘤浸润免疫细胞12239个。此外，本研究使用Chromium单细胞V(D)J试剂盒，对近10000个伴随有基因表达信息的T细胞进行了T细胞受体（T cell receptor, TCR）序列富集，由此可开展免疫细胞克隆性与转录表型多样性的相关研究。
实验整体设计如下：对外周血及肿瘤浸润免疫细胞开展5'端单细胞RNA测序；本研究采用10x Genomics 5'端基因表达分型试剂盒，对3例患者的免疫细胞开展单细胞RNA测序，并获取了T细胞的TCR序列信息。