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
Mouse samples were obtained from the PRBB (Barcelona Biomedical Research Park) Animal Facility. The samples were collected from both adult and embryonic mice of the C57BL/6 strain. Adult mice were selected based on standard laboratory guidelines, and embryos were harvested at the desired developmental stages under sterile conditions. All procedures were performed in accordance with institutional animal care and ethical guidelines to ensure the welfare of the animals. Total RNA Extraction Protocol: Total RNA samples were extracted from tissues or cell lines using the Thermo Fisher PureLink RNA Mini Kit. This kit utilizes a silica membrane-based purification system, which allows for the efficient isolation of high-quality RNA by binding nucleic acids to a silica membrane. Samples are lysed (using Trizol) and homogenized, and RNA is selectively bound to the membrane while impurities are washed away. The final purified RNA is eluted in RNase-free water, ensuring it is suitable for downstream applications. 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.