Long-read nascent RNA sequencing from S. pombe. Long-read nascent RNA sequencing from S. pombe

Long-read SMRT cDNA sequencing of nascent RNA from exponentially growing S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3’ end was labeled with a 3’ DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a transcriptome-wide PCR. SMRT DNA sequencing libraries were prepared subsequently. Overall design: Nascent RNA profiles were generated with long-read SMRT cDNA sequencing.

本研究采用指数生长期粟酒裂殖酵母（S. pombe）细胞的新生RNA开展长读长单分子实时（SMRT）cDNA测序，以获取单条转录本的转录延伸与剪接信息。新生RNA从该粟酒裂殖酵母的染色质组分中提取（Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010）。通过连接反应，使用3' DNA接头对新生RNA的3'端进行标记；该接头序列可作为全长逆转录的模板，随后通过全转录组范围的PCR扩增获得双链cDNA。后续完成SMRT DNA测序文库的构建。实验整体设计：通过长读长SMRT cDNA测序生成新生RNA的特征图谱。