Phelps Wesley A, Ayers Taylor N, Lee Miler T
Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA.
Methods Mol Biol. 2025;2923:163-180. doi: 10.1007/978-1-0716-4522-2_10.
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.
高通量RNA测序（RNA-seq）通常用于全转录组范围的基因表达定量分析。虽然该技术通常与聚腺苷酸（poly(A)）选择相结合，以富集信使RNA（mRNA），同时排除细胞中高度丰富的核糖体RNA（rRNA），但这种策略会使具有短或无poly(A)尾的mRNA定量不足，并且可能将poly(A)尾长度的变化与RNA水平的变化混为一谈。这在早期发育过程中尤为突出，因为在poly(A)+RNA-seq时间进程中，随着时间的推移母体mRNA的细胞质聚腺苷酸化可能会被误认为是基因组激活。在此，我们提出了一种使用可针对任何分类群定制的简化rRNA去除策略进行全RNA测序的方法。借助我们的Oligo-ASST网络工具设计反义DNA寡核苷酸，以稀疏覆盖rRNA的长度，这些寡核苷酸与热稳定核糖核酸酶H一起用于从总RNA样本中消化rRNA。经过柱净化后，富集mRNA的样本即可用于测序文库构建。
