Serial analysis of gene expression.

Serial analysis of gene expression (SAGE) is a method used to obtain comprehensive, unbiased and quantitative gene-expression profiles. Its major advantage over arrays is that it does not require a priori knowledge of the genes to be analyzed and reflects absolute mRNA levels. Since the original SAGE protocol was developed in a short-tag (10-bp) format, several modifications have been made to produce longer SAGE tags for more precise gene identification and to decrease the amount of starting material necessary. Several SAGE-like methods have also been developed for the genome-wide analysis of DNA copy-number changes and methylation patterns, chromatin structure and transcription factor targets. In this protocol, we describe the 17-bp longSAGE method for transcriptome profiling optimized for a small amount of starting material. The generation of such libraries can be completed in 7-10 d, whereas sequencing and data analysis require an additional 2-3 wk.