Ligate tags SAGE: Procedure Digest with “Tagging enzyme” BsmFI tm Isolate mRNA, RT to cDNA Digest with “Anchoring.

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Ligate tags SAGE: Procedure Digest with “Tagging enzyme” BsmFI tm Isolate mRNA, RT to cDNA Digest with “Anchoring enzyme” NlaIII Sequence

Mapping SAGE tags to genes AAAAAAA

Tags Position of NlaIII site

Experimental SAGE tags EST DataNo EST Data Transcripts with EST support Transcripts without EST support Conceptual mRNAs Map tags Predicted Gene ModelsGenomic DNA Sequence Theoretical SAGE tags Digest in silico Adjust 3’ UTRs UTR length distribution

Mapping SAGE tags to genes AAAAAAA

Internal polyA tracts AAAAAAA

NlaIII NlaIII Partial Digestion

Alternative Splicing AAAAAAA

Serial Analysis of Gene Expression How many genes can we identify with SAGE? How many have multiple tags? How many extra tags are due to internal polyAs? How many extra tags are due to NlaIII partial digestion Do genes with >1 tag have higher expression levels?

1.Extract theoretical SAGE tags from the virtual transcriptome -- make a note of which ones are upstream of a polyA tract 2.Identify which experimental SAGE tags could be due to partial digestion with NlaIII 3.Identify genes with multiple SAGE tags, subtract tags encountered in steps 1 and 2 4.Calculate the tag abundance for genes with just one tag and genes with multiple tags

1.The virtual transcriptome in a fasta file 2.A summary of tag abundance and tag-to- gene mapping from the website 3.A summary of tags that map to intron of known genes Starting material: