RNA-seq analysis of liquid-induced suspended animation (LISA) in C. elegans
To elucidate the molecular landscape associated with liquid-induced suspended animation (LISA) in C. elegans, we performed transcriptomic profiling of animals subjected to high-density isosmotic liquid conditions. Wild-type N2 animals were maintained at 20 C. To induce LISA, synchronized L4 larvae were incubated under static high-density liquid conditions (standing treatment) for 12 hours, while control animals were maintained under standard plate culture conditions prior to collection.At the time of sample collection, animals were washed off NGM plates using M9 buffer, collected into 1.5 mL microcentrifuge tubes, and homogenized using a tissue disruptor. Total RNA was extracted using the FastPure Cell-Tissue Total RNA Isolation Kit V2 (RC112-01, Vazyme) according to the manufacturer's instructions. RNA concentration and purity were measured using a NanoDrop spectrophotometer, and RNA integrity was assessed by agarose gel electrophoresis. For each sample, 1 ug of total RNA was used for library preparation and sequencing. Three biological replicates were included for each condition.Transcriptomic analysis revealed robust induction of stress-responsive genes during LISA. Notably, members of the small heat shock protein family, particularly the hsp-16 gene cluster, were among the most strongly upregulated transcripts. This induction was sharply confined to the genomic hsp-16 cluster without affecting adjacent genes. These findings define a distinct LISA-associated transcriptional state characterized by coordinated stress-responsive gene expression, consistent with a dormant yet physiologically primed condition.