REMC Standards and Guidelines for RNA-sequencing

Next-generation sequence based transcriptome methodologies (broadly referred to as RNA-seq) were initially developed in 2007 for massively parallel short read sequencing platforms. RNA-seq involves purification of RNA, followed by either selection of poly-A(+) RNA or depletion of ribosomal RNA. RNA is then converted into cDNA by one of two methods; 1) random priming, followed by cDNA fragmentation, end-repair and Illumina/SOLiD linker ligation or, 2) Enzymatic or chemical RNA fragmentation followed by linker ligation and cDNA generation. Following PCR amplification of tailed cDNA fragments with primers suitable for solid phase (Illumina) or emPCR (SOLiD) clonal amplification RNA-seq libraries are subjected to sequencing. Sequence alignment software is then used to compare the short sequence reads to reference genome and transcriptome databases, and exon-exon junction databases. From this analysis paradigm emerges data that is used for a variety of purposes, including the measurement of genelevel and exon-level expression abundance; detection of base changes (mutations and polymorphisms) relative to reference datasets; measurement of alternative splicing events; identification of gene fusion events; and identification of RNA editing events.