Transcriptome-wide measurement of poly(A) tail length and composition at subnanogram total RNA sensitivity by...

Poly(A) tails are added to the 3' ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Measuring poly(A) tails is critical for understanding their regulatory roles in almost every aspect of biological and medical studies. Previous methods for analyzing poly(A) tails require large amounts of input RNA (microgram-level total RNA), which limits their application. We recently developed a poly(A) inclusive full-length RNA isoform-sequencing method (PAIso-seq) at single-oocyte-level sensitivity (a single mammalian oocyte contains ~0.5 ng of total RNA) based on PacBio sequencing that enabled accurate measurement of the poly(A) tail length and non-A residues within the body of poly(A) tails along with the full-length cDNA, providing the opportunity to study precious in vivo samples with very limited input material. Here, we describe a detailed protocol for PAIso-seq library preparation from single mouse oocytes or bulk oocyte samples. In addition, we provide a complete bioinformatic pipeline to perform the analysis from the raw data to downstream analysis. The minimum time required is ~14.5 h for PAIso-seq double-stranded cDNA preparation, 2 d for PacBio sequencing in HiFi mode and 8 h for the initial data analysis.