Single cell RNA sequencing of skin T cells from patient with mycosis fungoides. Single cell RNA sequencing of skin T cells from patient with mycosis fungoides

Capture and processing of single skin T cells from patient with mycosis fungoides was performed using the Fluidigm C1 Autoprep system. Cells were loaded at a concentration of 2,000 cells/μL onto C1 integrated fluidic circuits (IFC) chips for 5-10 μm cells. All C1 capture sites were microscopically inspected to determine the sites containing only a single cell. Empty sites and those with multiple cells were excluded from further analysis. ERCC (External RNA Controls Consortium) spike-in RNAs were added and served as a control. SMARTer Ultra Low RNA Kit (Clontech) was used for reverse transcription and cDNA preamplification. The cDNA products from each cell were then used to prepare Illumina sequencing libraries and then sequenced on Illumina HiSeq2500 using single-end 100-base reads. Gene expression was quantified using Kallisto and then normalized to TPM values Overall design: single-cell RNA-seq investigation of a cutaneous T-cell lymphoma

本研究采用Fluidigm C1自动制备系统，对蕈样肉芽肿（mycosis fungoides）患者皮肤来源的单个T细胞开展捕获与处理。以2000个细胞/微升的浓度将细胞接种至适配5-10μm细胞的C1集成流体回路（integrated fluidic circuits, IFC）芯片中。通过显微镜检查所有C1捕获位点，筛选仅包含单个细胞的位点；空位点以及含有多个细胞的位点均被排除，不进行后续分析。添加外源RNA对照联盟（External RNA Controls Consortium, ERCC）的外参RNA作为对照。使用SMARTer超低总量RNA试剂盒（Clontech）完成反转录与cDNA预扩增。将每个细胞的cDNA产物用于构建Illumina测序文库，随后在Illumina HiSeq2500平台上采用单端100碱基读长进行测序。采用Kallisto进行基因表达定量，并将结果归一化为TPM值。整体实验设计：针对皮肤T细胞淋巴瘤的单细胞RNA测序研究。