Supplementary Information for:

Supplementary Methods Paired end ditags (PETs) Gene identification signature (GIS) analysis is a sequencing and mapping strategy that allows for the high-throughput demarcation of gene transcription boundaries, i.e. the 5’ and 3’ gene termini (Ng, et al., 2005). The GIS analysis procedure that produced the data we analyzed started with the isolation of polyA+ RNA from cells lines subject to different treatments: 1) the log phase of MCF7 cells, 2) MCF7 cells treated with estrogen (10nM beta-estradiol) for 12 hours, 3) HCT116 cells treated with 5FU (5-fluorouracil) for 6 hours, 4) the log phase of embryonic stem cell hES3 in feeder free culture condition. Full-length cDNAs (flcDNA) were generated from RNA and selected using the biotynlated CAP trapper method (Carninci, et al., 1996). The CAP trapper method relies on the introduction of a biotin group to the cap structure found at the 5’ end of full length mRNAs followed by first strand cDNA synthesis. Biotin residues are selected using streptavidin-coated magnetic beads, which results in the retention of only flcDNAs. BamHI and MmeI restriction sites are ligated to the 5’ and 3’ termini of the flcDNAs, which are then cloned to produce the GIS-flcDNA library. This library is digested with MmeI to yielding 18bp sequence fragments (signatures) from the 5’ and 3’ ends of flcDNAs. The 3’ end of the signature includes two A residues from the polyA tail. The 5’ and 3’ flcDNA signatures are covalently ligated to form 36bp paired-end ditags (PETs), each of which represents an individual transcript. PETs are exised using BamHI digestion and then concatenated and cloned for high-throughput sequencing. A single sequencing read of ~700bp leads, on average, to the characterization of 15 distinct PETs. The GIS cloning and sequence analysis resulted in 584,624 PETs for the log phase MCF7 cells, 153,179 PETs for the estrogen-treated MCF7 cells, 280,340 PETs for the HCT116 cells, and 1,799,970 PETs for the hES3 cells. These PETs were then mapped to the human genome using the following criteria: paired 5’ and 3’ ends must be on the same chromosome, they must be in the correct 5’-to-3’ order and orientation, they must be within 1 million base pairs, there must be a 16bp contiguous sequence match (out of 18bp) for the 5’ end of the PET and a 14bp contiguous match (out of 16bp) for the 3’ end of the PET. Using these criteria, most of the PET sequences (>90%) mapped to single locations in the human genome, but PETs mapping to 2-10 locations were also included in the analysis. The quality and mapping specificity of PETs has been confirmed in a number of different ways (Ng, et al., 2005). For instance, >95% of PETs map to known human gene transcripts and the vast majority fell within 10bp of the transcription start and termination sites. Most relevant to our study is the fact that the GIS analysis has been shown to be 30 times more efficient than standard cDNA methods for characterizing transcript and has resulted in the discovery of numerous previously uncharacterized transcripts. Thus, GIS is particularly suited to the discovery of alternative transcripts in the human genome of the kind initiated by ERV sequences.