Stem cell transcriptome profiling via massive-scale mRNA sequencing

Application of next-generation sequencing using the ABI SOLiD technology to mammalian transcriptome analysis enabled a survey of the content, the complexity and the developmental dynamics of the embryonic stem cell transcriptome in the mouse. Also in this issue, Mortazavi et al . report Illumina technology–based RNA-Seq analysis of the mouse transcriptome in three different tissues. We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A) + transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.