Strand-Specific RNA Sequencing for wild type and Drpd3 yeast entering quiescence [RNA-seq]

Strand-specific RNA sequencing was performed on wild type yeast in log phase, after diauxic shift, and after entry into quiescence with incorporation of external ERCC RNA spike-in controls to account for global changes in RNA abundance. We find that RNA profiles undergo at least two transitions: 1) From log-to-diauxic shift where stress response genes are induced and translational machinery is massively repressed. 2) From diauxic shift-to-quiescence, where global transcript abundance is repressed 15-fold. The transition from diauxic shift to quiescence was found to require Rpd3, as deletion of Rpd3 prevented the global repression of the transcriptome after the diauxic shift. Strand-specific RNA sequencing was carried out in biological replicate with external controls then transcript abundance was normalized to reflect RNA content per cell.

本研究对处于对数生长期、二次生长转换（diauxic shift）后以及进入静止期（quiescence）的野生型酵母开展了链特异性RNA测序（strand-specific RNA sequencing），并引入外部ERCC RNA spike-in对照（ERCC RNA spike-in control）以校正RNA丰度的整体变化。研究发现，酵母转录组谱至少经历两次显著转变：
1) 从对数生长期到二次生长转换阶段，此时应激反应基因被诱导表达，翻译相关分子机器被大规模抑制；
2) 从二次生长转换阶段到静止期阶段，此时全局转录本丰度被抑制至原水平的1/15。进一步研究表明，从二次生长转换到静止期的转变过程依赖Rpd3蛋白，因为敲除Rpd3基因会阻断二次生长转换后转录组的全局抑制现象。本研究通过生物学重复实验完成链特异性RNA测序，并借助外部对照对转录本丰度进行标准化，以反映单个细胞的RNA含量。