Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, CA, USA.
Sci Rep. 2013;3:1740. doi: 10.1038/srep01740.
We developed a novel Designed Primer-based RNA-sequencing strategy (DP-seq) that uses a defined set of heptamer primers to amplify the majority of expressed transcripts from limiting amounts of mRNA, while preserving their relative abundance. Our strategy reproducibly yielded high levels of amplification from as low as 50 picograms of mRNA while offering a dynamic range of over five orders of magnitude in RNA concentrations. We also demonstrated the potential of DP-seq to selectively suppress the amplification of the highly expressing ribosomal transcripts by more than 70% in our sequencing library. Using lineage segregation in embryonic stem cell cultures as a model of early mammalian embryogenesis, DP-seq revealed novel sets of low abundant transcripts, some corresponding to the identity of cellular progeny before they arise, reflecting the specification of cell fate prior to actual germ layer segregation.
我们开发了一种新颖的基于设计引物的 RNA 测序策略（DP-seq），该策略使用一组定义的七聚体引物来扩增来自有限量 mRNA 的大多数表达转录本，同时保留其相对丰度。我们的策略能够在低至 50 皮克 mRNA 的情况下进行高重复性扩增，同时在 RNA 浓度方面提供超过五个数量级的动态范围。我们还证明了 DP-seq 能够选择性地抑制我们测序文库中高度表达的核糖体转录本的扩增超过 70%。利用胚胎干细胞培养中的谱系分离作为早期哺乳动物胚胎发生的模型，DP-seq 揭示了一组新的低丰度转录本，其中一些对应于细胞后代在出现之前的身份，反映了在实际胚层分离之前细胞命运的特化。
