Serial Analysis of Gene Expression (SAGE): Detailed Protocol for Generating SAGE Catalogs of Mammalian Cell Transcriptomes

Abstract

Serial analysis of gene expression (SAGE) is a sequencing-based technique that permits the simultaneous evaluation of thousands of transcripts in a single assay. The technique generates large numbers of short tags, originating from the last unique location of a restriction enzyme recognition site in a cDNA generated from a single transcript. DNA in aqueous solution can be precipitated by EtOH in the presence of sodium or ammonium acetate. In certain cases, an increased volume of ethanol or 10 min dry ice/ethanol incubation is required on the precipitation step, as indicated in the text. Terminate the reaction when the bromphenol blue stain in the loading buffer migrates to the bottom of the gel. The optimal dilution of the ditag ligation reaction products for large-scale PCR is considered based on 102-bp band brightness to background ratio and highest dilution ratio. Once the conditions have been optimized, one needs to perform a large-scale PCR with a fresh LoTE dilution of the selected ditag ligation product as a template. Perform the reaction as described in step 10, except use a Thermo-Fast 96-well detection plate. © 2006 Elsevier Inc. All rights reserved.