CiTnI TSS identification by experimental molecular genetics and by high-throughput 5′-RACE sequence analysis of minor transcripts present in normal tailbud embryo RNA. Panel (A) TSS mapping by experimental molecular genetics. The panel shows, on the second sequence line, CiTnI (Atlantic allele) DNA in the region −550 to −491, and beneath it the very similar sequence of the Pacific allele (dots indicate identitity, and note that the ATG-based coordinates are shifted by 2 nt). The topmost sequence line shows the 5′-terminal sequence of a ∼500 bp CiTnI-LacZ mRNA 5′-RACE product generated from embryos expressing construct CiTnI(−819/−383)nZ. Four independent 5′-RACE clones all gave the same sequence, as shown (arbitrary anchor/oligo(dG) sequences introduced during the 5′-RACE procedure have been trimmed). The 5′-end of all four products mapped precisely to CiTnI nucleotide −523 (Atlantic allele, corresponding to −521 Pacific allele). Panels (B and C) CiTnI TSS mapping by high-throughput sequencing of random-primed oligocapping 5′-RACE products from naturally occurring transcripts present in normal tailbud embryo RNA. The panels show the distribution of 5′-reads mapping uniquely to CiTnI (Pacific allele) region −550 to −490 (B) or region −900 to −1 (C). Panel B corresponds spatially to Panel A, with each histogram bar representing a single nucleotide position. The spatial relationships of Panels B and C are indicated by dashed lines. In Panel C each histogram bar represents a 10-nt bin. The genomic position mapped for each read corresponds to the first nucleotide of the read for non-trans-spliced reads (black bars), and the first nt following the 16-nt SL sequence for trans-spliced reads (white bars). Note the logarithmic scale of the read count axes; all positions/bins in the region with no bars shown had zero reads mapped, and the fact that several bins in Panel C contained a single mapped read is shown, arbitrarily, by the shortest black bars visible. All other bars shown represent two or more mapped reads. Exact numbers for peak counts of non-trans-spliced and trans-spliced reads are indicated.