1. So many RNAs from individual loci 6 3 5 9 8 2a 2b 2c 11 10 Start Stop 1b 1a 1 13 4 7 12

2. Enrichment of cap/non-cap RNAsin CAGE libraries • Most RNA (cDNA) ribosomal • Random primer on total RNA • But with cap-selection: 0.7% ~ 3% ribosomal RNAs • (unwanted bad libraries: up to 50% has been ribosomal RNA in bad libraries) Before enrichment After enrichment (cap-trap) Ribosomal RNA = 90% Ribosomal RNA = 0.7% = 30 fold rRNA (over capped RNA) = 0.0071 fold rRNA (over capped RNA) Capped RNA = 3%? Capped RNA = 98%? 30 = 4225 fold 0.007 If starting ribosomal RNA= 80%, capped RNA= 5%, capped RNA in library= 90%, ribosomal contamination = 3%  Calculated enrichment = 485 fold  MOST tags represent cap sites

3. Poly-A minus RNA CAGE (9-12.5 millions tags/library), K562 Encode cell line. Overlapping tags grouped in clusters Although prostate has more tags than nuclear RNA, the nuclear poly A- library contains many more clusters

4. Poly-A-minus tag annotatiton The nuclear RNA is enriched in RNA (often in introns) to be analyzed

5. Poly-A minus CAGE mostly nuclear, overlap introns and TSSs, cytoplasmic more on exons