Optimizing mRNA enrichment: a comparative analysis of strategies and guidance for improving efficiency

The pervasive presence of ribosomal RNA (rRNA) in total RNA poses a considerable challenge to the accurate analysis of cellular transcriptomes. Caused by rRNA contamination, low sequencing depth of non-rRNA species can lead to difficulties in their secondary structure mapping, using strategies coupled with NGS, such as SHAPE-seq, SHAPE-MaP, or DMS-seq. We comprehensively analyzed strategies for Saccharomyces cerevisiae mRNA enrichment. Here, we apply the NGS-based method to evaluate rRNA content in yeast RNA samples after two rounds of selected mRNA enrichment with oligo (dT)25 magnetic beads. In brief, yeast culture (Saccharomyces cerevisiae, strain BY4741) was treated with DMSO (a control sample in SHAPE experiments). Then, total RNA was extracted, followed by two rounds of mRNA enrichment using oligo (dT)25 magnetic beads (New England Biolabs). DNAse treatment was performed after the first round of mRNA enrichment. After the second round, RNA was reverse transcribed with random nonamer primers in Mn2+ conditions, and dsDNA was made by non-directional second-strand synthesis. Finally, samples were converted into sequencing libraries and sequenced in the PE150 on Illumina system.

核糖体RNA（rRNA）在总RNA中的广泛存在，给细胞转录组的精准分析带来了极大挑战。由rRNA污染导致的非rRNA物种测序深度不足，会使其二级结构绘图工作难以开展——此类研究常结合下一代测序（next-generation sequencing, NGS）策略，例如SHAPE-seq、SHAPE-MaP或DMS-seq。我们对酿酒酵母（Saccharomyces cerevisiae）的mRNA富集策略进行了全面分析。本研究采用基于NGS的方法，评估经两轮寡聚(dT)25磁珠富集筛选后的酵母RNA样本中的rRNA残留量。简言之，我们对酿酒酵母菌株BY4741的培养物进行二甲基亚砜（DMSO）处理（作为SHAPE实验的对照样本）。随后提取总RNA，先后使用两轮寡聚(dT)25磁珠（New England Biolabs）进行mRNA富集；第一轮富集完成后即进行DNA酶处理。第二轮富集结束后，在Mn²+条件下使用随机九聚体引物对RNA进行反转录，再通过无方向性的第二链合成制备双链DNA。最终将样本构建为测序文库，并在Illumina测序平台上采用PE150模式完成测序。