Long-read transcriptomics of a diverse human cohort reveals widespread ancestry bias in gene annotation
Long-read transcriptomics of a diverse human cohort reveals widespread ancestry bias in gene annotation
43 Lymphoblastoid cell lines (LCLs) were obtained from the Coriell Institute for Research, representing individuals from diverse ethnic backgrounds. Cells were cultured in RPMI complete medium supplemented with 15% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (final concentrations: 100 U/mL penicillin and 100 µg/mL streptomycin). Cells were maintained at 37°C in a 5% CO₂ incubator and collected at mid-log phase to ensure viability and consistency. 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.