PASP — a whole-transcriptome poly(A) tail length determination assay for the Illumina platform

The poly(A) tail, co-transcriptionally added to most eukaryotic mRNAs, plays an important role in posttranscriptional regulation through modulating mRNA stability and translational efficiency. The length of the poly(A) tail is dynamic, decreasing or increasing in response to various stimuli through the action of enzymatic complexes, and changes in tail length are exploited in regulatory pathways implicated in various biological processes. To date, assessment of poly(A) tail length has mostly relied on protocols targeting only a few transcripts. We present PASP (‘poly(A) tail sequencing protocol’), a whole-transcriptome approach to measure tail lengths — including a computational pipeline implementing all necessary analyses. PASP uses direct Illumina sequencing of cDNA fragments obtained through G-tailing of poly(A)-selected mRNA followed by fragmentation and reverse transcription. Analysis of reads corresponding to spike-in poly(A) tracts of known length indicated that mean tail lengths can be confidently measured, given sufficient coverage. We further explored the utility of our approach by comparing tail lengths estimated from wild type and ∆ccr4-1/pan2 mutant yeasts. The yeast whole-transcriptome tail length distributions showed high consistency between biological replicates, and the expected upward shift in tail lengths in the mutant samples was detected. This suggests that †Current address: Oxford Nanopore Technologies, Edmund Cartwright House, 4 Robert Robinson Avenue, Oxford Science Park, Oxford, OX4 4GA, United Kingdom. . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/060004 doi: bioRxiv preprint first posted online Jun. 21, 2016;