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High-throughput RNA sequencing (RNA-seq) is commonly used to quantify gene expression transcriptome-wide. While usually paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA) to the exclusion of highly abundant ribosomal RNA (rRNA) in the cell, this strategy will under-quantify mRNA with short or absent poly(A) tails and can conflate changes in poly(A) tail length with changes in RNA level. This is notably an issue during early development, when cytoplasmic polyadenylation of maternal mRNA over time can be mistaken for genome activation in poly(A) + RNA-seq time courses. Here, we present a method to perform total RNA-seq using a streamlined rRNA depletion strategy customizable to any taxon. Antisense DNA oligos are designed with the aid of our Oligo-ASST web tool to sparsely tile the length of the rRNA, which are used with thermostable RNaseH to digest rRNA from a total RNA sample. After column cleanup, the mRNA-enriched sample is ready for sequencing library construction.

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Ribosomal RNA Depletion for Poly(A)-Tail-Independent Quantification of Genome Activation

  • 实验方案

  • 发表日期：

    2025年

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  • Wesley A. Phelps,
  • Taylor N. Ayers,
  • Miler T. Lee

摘要

High-throughput RNA sequencing (RNA-seq) is commonly used to quantify gene expression transcriptome-wide. While usually paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA) to the exclusion of highly abundant ribosomal RNA (rRNA) in the cell, this strategy will under-quantify mRNA with short or absent poly(A) tails and can conflate changes in poly(A) tail length with changes in RNA level. This is notably an issue during early development, when cytoplasmic polyadenylation of maternal mRNA over time can be mistaken for genome activation in poly(A) + RNA-seq time courses. Here, we present a method to perform total RNA-seq using a streamlined rRNA depletion strategy customizable to any taxon. Antisense DNA oligos are designed with the aid of our Oligo-ASST web tool to sparsely tile the length of the rRNA, which are used with thermostable RNaseH to digest rRNA from a total RNA sample. After column cleanup, the mRNA-enriched sample is ready for sequencing library construction.