Efficient and specific DNA oligonucleotide rRNA probe-based rRNA removal in Talaromyces marneffei

Emerging evidence showed that lncRNAs play important roles in a wide range of biological processes of fungi such as Saccharomyces cerevisiae. However, systemic identification of lncRNAs in non-model fungi is a challenging task as the efficiency of rRNA removal has been proved to be affected by mismatches of universal rRNA-targeting probes of commercial kits, which forces deeper sequencing depth and increases costs. Here, we developed a low-cost and simple rRNA depletion method (rProbe) that could efficiently remove more than 99% rRNA in both yeast and mycelium samples of Talaromyces marneffei. The efficiency and robustness of rProbe were demonstrated to outperform the Illumina Ribo-Zero kit. Using rProbe RNA-seq, we identified 115 differentially expressed lncRNAs and constructed lncRNA-mRNA co-expression network related to dimorphic switch of T. marneffei. Our rRNA removal method has the potential to be a useful tool to explore non-coding transcriptomes of non-model fungi by adjusting rRNA probe sequences species specifically.