PAT-seq: a method to study the integration of 3′-UTR dynamics with gene expression in the eukaryotic transcriptome

Alternative cleavage and adenylation. (A) To validate PAT-seq data, gene-by-gene T₁₂VN-PAT and ePAT assays were performed. The T₁₂VN-PAT assays indicate the size of the PCR amplicons with a limiting (A12)-poly(A) tail whereas the ePAT assay includes the full-native poly(A) tail in amplicons. Note the up-shift in amplicons sizes in the Δccr4 mutant samples. (B) Schematic of the antiparallel orientation and the Ty3 LTR YORWsigma3 (LTRσ3) transcript and of SNF2. (C) APA shifts the transcript cleavage and adenylation between Proximal (P) and Distal (D) recognition sites. (D) The dinucleotide preceding the adenylation site is nonrandom. The flattened transcriptome indicates the percentage of dinucleotide usage at unique adenylation sites, comparing abundant and rare sites equally. The full transcriptome indicates all reads encompassing the adenylation site, incorporating transcript abundance.