Identification of tens of thousands of novel gene loci in the human and mouse genomes through targeted full-length long-read RNA sequencing
Identification of tens of thousands of novel gene loci in the human and mouse genomes through targeted full-length long-read RNA sequencing
Commercial samples obtained from Ambion (Thermo Fisher Scientific) or Clontech-Takara Bio: Both Ambion and Clontech-Takara Bio are brands specializing in life science research products, with a focus on RNA technologies. Ambion is a brand owned by Thermo Fisher Scientific, a global provider of scientific research products and services, while Clontech is part of Takara Bio, known for molecular biology tools. Therefore, \"commercial samples obtained from Ambion (Thermo Fisher Scientific) or Clontech-Takara Bio\" refers to RNA samples purchased from these brands for research purposes. Total RNA was extracted from the collected samples using protocols performed by commercial providers Ambion (Thermo Fisher Scientific) or Clontech-Takara Bio. Although the exact kit or extraction protocol used was not specified, the RNA extraction followed the manufacturers' standard procedures, ensuring high-quality and reliable RNA yield for downstream applications. Capture Long-Read Sequencing (CLS): The CLS library preparation protocol combines targeted RNA capture using streptavidin and biotin-based probes with third-generation long-read sequencing technologies like ONT and PacBio. This method allows for the preparation of sequencing libraries that can selectively enrich specific regions of the transcriptome, enabling the identification of novel transcripts and splice variants with high accuracy and coverage. CapTrap cDNA library preparation: CapTrap cDNA library preparation is a long-read sequencing protocol that selectively enriches 5'-capped transcripts by capturing the 5' caps of transcripts. This protocol uses Poly(A) tail selection to specifically target mRNA, and a 5' cap-specific oxidation reaction coupled to biotin-streptavidin to capture the 5' cap structure. The captured mRNA is then converted to cDNA and the library is prepared with 5' and 3' specific linkers to enrich for full-length libraries. CapTrap is particularly useful for studying transcription start sites, alternative splicing, and post-transcriptional modifications.