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.