Expression profiling of circular RNAs in cells from human carotid plaque biopsies by single-cell RNA sequencing

To identify the origin of circular RNAs (circRNAs) in human atherosclerotic plaques, we analyzed the expression levels of circRNAs identified during the differentiation of human pluripotent stem cells (hiPSCs) into vascular endothelial cells (ECs) and smooth muscle cells (SMCs) in carotid biopsies by single-cell RNA sequencing. Dissociated cells from human carotid atherosclerotic plaques (n = 15 subjects) were enriched for SMCs, ECs and immune cells and sorted by FACS. Single-cell cDNA libraries were prepared according to the Smart-Seq2 protocol using oligo(dT) primers and sequenced (PubMed ID: 38639096, GEO: GSE260657). For circRNA analyzes, raw reads were mapped to a sequence assembly consisting of GRCh37 and previously defined circRNAs (GEO: GSE210361) using segemehl, with circRNA contigs consisting of 100 nt at each site of each backsplicing event. Reads located on the backsplicing event were counted with featureCounts if the sequenced reads covered at least 8 nt of a 10 nt window located on the backsplicing event. Processed data are given as tab-delimited text files with raw counts for each circRNA and cell. For this third-party reanalysis, the reanalyzed samples were submitted to GEO previously in GSE260657. The table below lists the GEO accessions reused/reanalyzed for this study.

为明确人类动脉粥样硬化斑块中环状RNA（circular RNAs, circRNAs）的来源，我们通过单细胞RNA测序分析了人类多能干细胞（human pluripotent stem cells, hiPSCs）向血管内皮细胞（vascular endothelial cells, ECs）和平滑肌细胞（smooth muscle cells, SMCs）分化过程中鉴定出的circRNA在颈动脉活检样本中的表达水平。我们从15名受试者的人类颈动脉粥样硬化斑块中解离得到细胞，对其中的平滑肌细胞、内皮细胞及免疫细胞进行富集后，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）完成分选。依照Smart-Seq2实验流程，使用寡聚(dT)引物制备单细胞cDNA文库并进行测序（PubMed ID: 38639096，GEO: GSE260657）。针对环状RNA分析，我们使用segemehl工具将原始测序reads比对至由GRCh37基因组序列与此前已定义的环状RNA序列（GEO: GSE210361）组成的序列组装库，其中环状RNA重叠群（circRNA contigs）在每个反向剪接事件的两侧各包含100 nt序列。若测序reads覆盖反向剪接事件所在10 nt窗口中至少8 nt，则使用featureCounts对该反向剪接事件上的reads进行计数。处理后的数据以制表符分隔的文本文件形式提供，包含每个环状RNA与每个细胞的原始计数。本研究为第三方重分析，原分析样本此前已提交至GEO数据库的GSE260657条目。下表列出了本研究中复用/重分析的GEO登录号。