Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines

Abstract

We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of ∼5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.[Supplemental material is available online at www.genome.org.]Serial analysis of gene expression (SAGE) offers a particularly attractive technology for profiling eukaryotic transcriptomes (Velculescu et al. 1995) because of the digital and quantitative nature of the data, its efficient sampling of short sequence tags from known and novel mRNA transcripts, and its theoretically unlimited dynamic range. Numerous improvements to the original technology have been described (Peters et al. 1999;Saha et al. 2002;Gowda et al. 2004;Heidenblut et al. 2004;Wei et al. 2004;Kodzius et al. 2006). These include the production of longer tags, which have improved the specificity of tag-to-gene mapping (Saha et al. 2002;Matsumura et al. 2003), and modifications designed to facilitate library construction from nanogram quantities of total RNA (Peters et al. 1999;Neilson et al. 2000). Recently, the use of SAGE-like procedures to identify regions of the genome interacting with DNA-binding proteins has been described (Impey et al. 2004;Chen and Sadowski 2005;Kim et al. 2005;Loh et al. 2006;Wei et al. 2006). Such approaches represent viable alternatives to ChIP-on-chip (Ren et al. 2000).SAGE is among the few relatively accessible digital gene expression profiling technologies capable of generating comprehensive transcriptome profiles. Nevertheless, challenges associated with laborious library construction and generally limited access to inexpensive automated DNA sequencing have restricted its application to large-scale initiatives. Experiments at our Genome Center ) and e...

Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential

“…The cDNA was purified on a Chroma-Spin 200 Column (BD), and its concentration determined using a spectrophotometer (GeneQuant Pro; Biochrom, Cambridge, United Kingdom). The amplified cDNA was then processed according to a modified version of the standard LongSAGE protocol using the I-SAGE Long kit (Invitrogen, Carlsbad, CA) as described in detail in Khattra et al 25 After analysis of data quality from a first 384-well sequencing plate, each library was sequenced to a sampling depth of 44 506 (adult BM) and 200 319 (FL) raw tags (GEO Series accession no. GSE13243; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc ϭ GSE13243).…”

“…25 In brief, RNA was reverse-transcribed, and the cDNA obtained was amplified using the switching mechanism at the 5Ј end of RNA transcripts (SMART) cDNA amplification kit (BD) following the manufacturer's protocol, but using a modified template switching primer containing an AscI digestion site. The first strand cDNA was purified with a NucleoSpin column (Clontech Laboratories, Mountain View, CA) and then amplified using a modified PCR primer that contained a biotin molecule at its 5Ј end and the Advantage II PCR kit (BD).…”

Next-generation tag sequencing for cancer gene expression profiling

“…Tag-seq is a variant of LongSAGE as described (Siddiqui et al 2005;Khattra et al 2007), with modifications forgoing the requisite production of ditags and concatemers and allowing direct sequencing on the Illumina Genome Analyzer (Fig. 1).…”

The transcription start site landscape of C. elegans

“…We therefore examined the association between each motif and the expression of its associated gene (defined as the nearest downstream gene). As a measure of tissue-specific expression, we used the publicly available LongSAGE data, a comprehensive collection of SAGE data derived from a variety of tissues (e.g., oocytes, embryos, gut, muscle, hypodermal, neurons, neural cells, pharynx cells, and gonad: British Columbia C. elegans Gene Expression Consortium, http://elegans.bcgsc.bc.ca/) ( Jones et al 2001;McKay et al 2003;Blacque et al 2005;Khattra et al 2007;McGhee et al 2007;Meissner et al 2009;Wang et al 2009). Two thousand three hundred and forty-two (2342) genes have SAGE data as well as at least one of our identified motifs upstream of their TSS.…”