Supplementary Table

Long-read sequencing has emerged as a powerful tool for uncovering novel transcripts and genes. However, existing protocols often lack confidence in identifying the transcription start site (TSS) and fail to capture non-poly(A) RNA, thereby limiting the discovery of novel genes, particularly long non-coding RNAs (lncRNAs). In this study, we introduce Cap-trap full-length cDNA sequencing (CFC-seq), a comprehensive protocol that combines Cap-trapping and poly(A)-tailing with Oxford Nanopore sequencing. This protocol enables precise identification of TSSs and full-length transcripts. Applying CFC-seq to two in vitro differentiation time courses resulted in approximately 236 million mappable reads. The transcript Start-site Aware Long-read Assembler (SALA) was developed for de novo assembling the transcript models, leading to the identification of 39,425 confident novel genes. Using this dataset, enhancer-derived ncRNAs were re-defined with longer length and more splicing activity, which were correlated with enhancer structure. Compared to enhancers with CpG islands, TATA box enhancers were shown to be more cell type specific with fewer chromatin interaction but produced longer and more stable polyadenylated RNA. A significant proportion of these TATA box-derived eRNAs originated from LTR transposable elements. Overall, this study systematically annotated ~24,000 novel eRNA genes and correlated their transcription properties with enhancer structure.长读长测序（Long-read sequencing）已成为发掘新型转录本与基因的强有力工具。然而，现有实验方案往往难以保证转录起始位点（transcription start site, TSS）鉴定的可信度，且无法捕获非多聚腺苷酸RNA，进而限制了新型基因的发掘，尤其是长链非编码RNA（long non-coding RNAs, lncRNAs）。本研究提出了帽捕获全长cDNA测序（Cap-trap full-length cDNA sequencing, CFC-seq）——一种将帽捕获、多聚腺苷酸尾加尾与牛津纳米孔测序（Oxford Nanopore sequencing）相结合的综合性实验方案，可精准鉴定转录起始位点与全长转录本。我们将CFC-seq应用于两组体外分化时间序列样本，共获得约2.36亿条可比对读段。研究团队开发了转录起始位点感知长读长组装器（transcript Start-site Aware Long-read Assembler, SALA）用于从头组装转录本模型，最终鉴定得到39425个高置信度新型基因。依托该数据集，研究人员重新定义了增强子来源非编码RNA：这类RNA具有更长的长度与更高的剪接活性，并与增强子结构存在显著关联。相较于携带CpG岛的增强子，携带TATA盒的增强子表现出更强的细胞类型特异性，其染色质相互作用更少，但可产生更长且更稳定的多聚腺苷酸化RNA。其中，相当比例的TATA盒来源增强子RNA（enhancer RNA, eRNA）源自长末端重复序列转座因子（long terminal repeat transposable elements, LTR）。总体而言，本研究系统性注释了约24000个新型增强子RNA基因，并将其转录特性与增强子结构进行了关联分析。