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Three-dimensional (3D) spheroid models are crucial for cancer research, offering more accurate insights into tumour biology and drug responses than traditional 2D cell cultures. However, inconsistent and low-throughput spheroid production has hindered their application in drug screening. Here, we present an automated high-throughput platform for a spheroid selection, fabrication, and sorting system (SFSS) to produce uniform gelatine-encapsulated spheroids (GESs) with high efficiency. SFSS integrates advanced imaging, analysis, photo-triggered fabrication, and microfluidic sorting to precisely control spheroid size, shape, and viability. Our data demonstrate that our SFSS can produce over 50 GESs with consistent size and circularity in 30 min with over 97% sorting accuracy while maintaining cell viability and structural integrity. We demonstrated that the GESs can be used for drug screening and potentially for various assays. Thus, the SFSS could significantly enhance the efficiency of generating uniform spheroids, facilitating their application in drug development to investigate complex biological systems and drug responses in a more physiologically relevant context. [ABSTRACT FROM AUTHOR]

Characterization of the subcellular distribution of RNA is essential for understanding the molecular basis of biological processes. Here, the subcellular nanopore direct RNA‐sequencing (DRS) of four lung cancer cell lines (A549, H1975, H358, and HCC4006) is performed, coupled with a computational pipeline, Low‐abundance Aware Full‐length Isoform clusTEr (LAFITE), to comprehensively analyze the full‐length cytoplasmic and nuclear transcriptome. Using additional DRS and orthogonal data sets, it is shown that LAFITE outperforms current methods for detecting full‐length transcripts, particularly for low‐abundance isoforms that are usually overlooked due to poor read coverage. Experimental validation of six novel isoforms exclusively identified by LAFITE further confirms the reliability of this pipeline. By applying LAFITE to subcellular DRS data, the complexity of the nuclear transcriptome is revealed in terms of isoform diversity, 3'‐UTR usage, m6A modification patterns, and intron retention. Overall, LAFITE provides enhanced full‐length isoform identification and enables a high‐resolution view of the RNA landscape at the isoform level. [ABSTRACT FROM AUTHOR]