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.
In the present study, we evaluated the physicochemical and microbial properties, microbial diversity, and volatile organic compounds (VOCs) of Jinhua ham prepared using Taihu black pigs during a ripening period of 210 days. During the eight timepoints, the protein oxidation, proteolysis indexes, and TBARS values of Jinhua ham had different change trends. The growth of total bacteria and total Staphylococcus showed a decline, while the growth trend of total fungi was parabolic. Staphylococcus equorum was the dominant bacterial species, and the relative abundance were 19.3% (day 180)-76.7% (day 120). Debaryomyces hansenii and Eurotium sp. were the dominant fungus species, with opposite change trends. GC–IMS analysis detected 84 volatile compounds, with alcohols and aldehydes being the dominant volatile compounds in the late ripening period. The correlation analysis between microbial diversity and VOCs showed that Staphylococcus equorum was significantly negatively correlated with eight VOCs, while Debaryomyces hansenii and Eurotium sp. were significantly correlated with 31 and 29 VOCs respectively. Our findings reveal the quality, microbial diversity at species level, volatile compound characteristics, and relationship between microbial communities and flavor formation of Jinhua ham prepared using Taihu black pigs.
危险品化学品经营许可证（带存储）