Owens Nick D L, De Domenico Elena, Gilchrist Michael J
The Francis Crick Institute, NW1 1ST London, United Kingdom.
The Francis Crick Institute, NW1 1ST London, United Kingdom
Cold Spring Harb Protoc. 2019 Jun 3;2019(6):pdb.prot098368. doi: 10.1101/pdb.prot098368.
Here we consider RNA-Seq, used to measure global gene expression through RNA fragmentation, capture, sequencing, and subsequent computational analysis. , with its large number of RNA-rich, synchronously developing, and accessible embryos, is an excellent model organism for exploiting the power of high-throughput sequencing to understand gene expression during development. Here we present a standard RNA-Seq protocol for performing two-state differential gene expression analysis (between groups of replicates of control and treated embryos) using Illumina sequencing. Samples contain multiple whole embryos, and polyadenylated mRNA is measured under relative normalization. The protocol is divided into two parts: wet-lab processes to prepare samples for sequencing and downstream computational analysis including quality control, quantification of gene expression, and differential expression.
在这里，我们考虑RNA测序，它通过RNA片段化、捕获、测序以及后续的计算分析来测量整体基因表达。由于其拥有大量富含RNA、同步发育且易于获取的胚胎，是利用高通量测序的强大功能来理解发育过程中基因表达的优秀模式生物。在这里，我们展示了一种标准的RNA测序方案，用于使用Illumina测序进行双态差异基因表达分析（在对照胚胎和处理胚胎的重复组之间）。样本包含多个完整胚胎，并在相对标准化的条件下测量多聚腺苷酸化mRNA。该方案分为两个部分：用于制备测序样本的湿实验过程以及包括质量控制、基因表达定量和差异表达分析在内的下游计算分析。
