Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

Genomes are pervasively transcribed leading to stable and unstable transcripts that define functional regions of genomes and contribute to cellular phenotypes. Defining comprehensive nascent transcriptomes is pivotal to understand gene regulation, disease processes, and the impact of extracellular signals on cells. However, currently employed methods are laborious, technically challenging and costly. We developed single-nucleotide resolution 4sU-sequencing (SNU-Seq), involving pulse labelling, biotinylation and direct isolation of nascent transcripts. Artificial poly-(A)-tailing of the 3’ most nucleotide of nascent transcripts ensures oligo-d(T) primer-based library preparation and sequencing using commercial 3’ RNA-Seq kits. We show that SNU-Seq is a cost-effective new method generating even read profiles across transcription units. We used SNU-Seq to identify transcription elongation parameters, to map usage of polyadenylation (PAS) sites and novel enhancers. Remarkably, 4sU labelled nascent RNA accumulates short ∼100nt transcripts that map to the 5’ end of genes. We show that isolation of these short nascent RNA and sequencing the 5’ and 3’ ends using size-selected SNU-Seq (ssSNU-Seq) provides highly sensitive annotations of mapped and novel TSSs, promoter-proximal pause/termination sites. Thus, SNU-seq and ssSNU-seq combined yield comprehensive transcriptomics data at low cost with high spatial and temporal resolution. Highlights - SNU-Seq maps nascent transcripts at base-pair resolution, with high sensitivity and low cost - SNU-Seq detects comprehensive polyadenylation sites. - SNU-Seq maps the promoter proximal pause 60-80 nt from the TSS. - Size-selected SNU-Seq yields highly sensitive and novel TSS annotations

基因组普遍被转录，产生稳定和不稳定的转录本，这些转录本界定了基因组的功能区域，并有助于细胞表型的形成。确定全面的新生转录组对于理解基因调控、疾病过程以及细胞外信号对细胞的影响至关重要。然而，目前所采用的方法费力、技术上具有挑战性且成本高昂。我们开发了单核苷酸分辨率的4sU测序（SNU - Seq），包括脉冲标记、生物素化以及新生转录本的直接分离。对新生转录本3’末端核苷酸进行人工多聚腺苷酸化确保了基于寡聚d(T)引物的文库制备以及使用商业3’ RNA - Seq试剂盒进行测序。我们表明SNU - Seq是一种具有成本效益的新方法，能在转录单元上产生均匀的读数分布。我们利用SNU - Seq来确定转录延伸参数，绘制多聚腺苷酸化（PAS）位点的使用情况以及新的增强子图谱。值得注意的是，4sU标记的新生RNA积累了约100nt的短转录本，这些转录本定位在基因的5’端。我们表明分离这些短的新生RNA，并使用大小选择的SNU - Seq（ssSNU - Seq）对5’和3’端进行测序，能够对已定位的和新的转录起始位点（TSSs）以及启动子近端暂停/终止位点进行高度灵敏的注释。因此，SNU - Seq和ssSNU - Seq相结合能够以低成本产生具有高时空分辨率的全面转录组学数据。 亮点： - SNU - Seq以碱基对分辨率绘制新生转录本图谱，具有高灵敏度和低成本的特点。 - SNU - Seq能检测全面的多聚腺苷酸化位点。 - SNU - Seq绘制出距离转录起始位点（TSS）60 - 80nt的启动子近端暂停位点。 - 大小选择的SNU - Seq能产生高度灵敏和新的转录起始位点注释。