Introduction to RNA Sequencing and Analysis

RNA sequencing (RNA-Seq) uses the capabilities of high-throughput sequencing methods to provide insight into the transcriptome of a cell. Compared to previous Sanger sequencing- and microarray-based methods, RNA-Seq provides far higher coverage and greater resolution of the dynamic nature of the transcriptome. Beyond quantifying gene expression, the data generated by RNA-Seq facilitate the discovery of novel transcripts, identification of alternatively spliced genes, and detection of allele-specific expression. Recent advances in the RNA-Seq workflow, from sample preparation to library construction to data analysis, have enabled researchers to further elucidate the functional complexity of the transcription. In addition to polyadenylated messenger RNA (mRNA) transcripts, RNA-Seq can be applied to investigate different populations of RNA, including total RNA, pre-mRNA, and noncoding RNA, such as microRNA and long ncRNA.

The introduction of high-throughput next-generation sequencing (NGS) technologies revolutionized transcriptomics. This technological development eliminated many challenges posed by hybridizationbased microarrays and Sanger sequencing-based approaches that were previously used for measuring gene expression. A typical RNA-Seq experiment consists of isolating RNA, converting it to complementary DNA (cDNA), preparing the sequencing library, and sequencing it on an NGS platform…(read more)


Overview of RNA-Seq. First, RNA is extracted from the biological material of choice (e.g., cells, tissues).
Second, subsets of RNA molecules are isolated using a specific protocol, such as the poly-A selection protocol to
enrich for polyadenylated transcripts or a ribo-depletion protocol to remove ribosomal RNAs. Next, the RNA is
converted to complementary DNA (cDNA) by reverse transcription and sequencing adaptors are ligated to the
ends of the cDNA fragments. Following amplification by PCR, the RNA-Seq library is ready for sequencing.

Kukurba KR, Montgomery SB. (2015) RNA Sequencing and Analysis. Cold Spring Harb Protoc [Epub ahead of print]. [abstract]

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