An Easy, Cost‐Effective, and Scalable Method to Deplete Human Ribosomal RNA for RNA‐seq

RNA sequencing (RNA-seq) is a powerful and increasingly prevalent method to characterize and quantify the transcriptome. Ribosomes are extremely abundant, however, and approximately 80% of total RNA is ribosomal RNA (rRNA). Therefore, to detect and quantify less abundant yet biologically important transcripts such as messenger RNA (mRNA) and long noncoding RNAs (lncRNA), it is essential to minimize the rRNA being sequenced. Although commercial methods exist to deplete rRNA from total RNA samples before sequencing, they are expensive and require specific amounts of input RNA, and the most commonly used kit is no longer available as a stand-alone product. Here, we present an optimized rRNA depletion protocol using RNase H and DNA oligonucleotides complementary to human rRNA transcripts. This protocol includes guidelines for DNA oligo preparation, RNA:DNA hybridization, RNase H cleavage and RNA cleanup, and benchmarking of rRNA depletion. The method is flexible because the user can include additional complementary DNA oligos directed against any abundant transcript in their particular system. Furthermore, the performance of this rRNA depletion approach is comparable to or better than that of commercial kits, at a fraction of the cost and across a wide range of input RNA amounts. © 2021 Wiley Periodicals LLC. Basic Protocol: Specific depletion of rRNA transcripts from human total RNA Support Protocol: Preparation of the rRNA depletion DNA oligonucleotide pool.