title: Long-read transcriptomics of a diverse human cohort reveals widespread ancestry bias in gene annotation
design: Long-read transcriptomics of a diverse human cohort reveals widespread ancestry bias in gene annotation
library_construction_protocol: 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.