This study provides a comprehensive benchmark of long-read RNA sequencing (lrRNA-seq) across bulk and single-cell platforms—Illumina, ONT, and PacBio (PB)—using a Fragile X syndrome cellular model. The authors reveal specific platform biases: bulk PB under-detects short transcripts (<1.25 kb), bulk ONT misses long transcripts (>5 kb), and single-cell platforms frequently capture truncated transcripts. Among the evaluated quantification tools, Isosceles, Miniquant, and Oarfish offered the best balance of computational efficiency and accuracy for downstream tasks. Additionally, the study highlights that single-cell PB sequencing requires three to four times greater depth than bulk sequencing to achieve comparable statistical power for transcript discovery and differential expression analysis, ultimately offering practical guidelines for future transcriptomic study designs.