Catalogue of mitotic chromosome-associated small RNAs in mouse 3T3 cell line

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Mitotic chromosomes are essential for faithful genome segregation and play a central role in ensuring genomic stability and the proper generation of daughter cells (Santaguida et al., 2017, Batty andGerlich, 2019). Nearly two centuries have passed since the initial discovery of chromosomes (Schulz-Schaeffer, 1980) and extensive research has since revealed that mitotic chromosomes are supramolecular complexes of DNA-protein-RNA, containing not only DNA and diverse proteins but also a significant amount of RNAs (Hearst and Botchan, 1970, Uchiyama et al., 2005, Ohta et al., 2010, Meng et al., 2016, Shen et al., 2022, Zhang et al., 2023, Zhang et al., 2024). Mitotic chromosome-associated proteins have been extensively studied using various isolation methods, leading to the identification of thousands of proteins that can be classified into distinct functional categories (Uchiyama et al., 2005, Ohta et al., 2010). Although studies on mitotic chromosome-associated RNAs (mCARs) remain limited, notable progress has been made in recent years. Several studies have demonstrated that certain mCARs play important regulatory roles during mitosis (Rosic et al., 2014, Ma et al., 2022). Our group was the first to perform sequencing-based identification of mCARs using 5'-tag sequencing, leading to the discovery of over one thousand mCARs (Meng et al., 2016). Our follow-up study provides a comprehensive and quantitative catalog of full-length mCARs across multiple species and cell types using highdepth RNA sequencing. This analysis identified a large number of mCARs species, uncovering thousands of enriched and previously unannotated transcripts (Zhang et al., 2024). Another group using a crude isolation method to obtain mCARs also showed that many RNAs are stably maintained and highly abundant on mitotic chromosomes (Shen et al., 2022).However, the investigation of mitotic chromosome-associated small RNAs (mCASRs) remains lacking. Although previous studies have identified a large number of RNAs that are tightly bound to mitotic chromosomes, they have primarily focused on long RNAs due to limitations in library preparation methods (Meng et al., 2016, Shen et al., 2022, Zhang et al., 2024), thereby resulting in the underrepresentation of small RNAs in these data. Yet small RNAs have diverse subcellular localizations (e.g. cytosol, nuclei) and numerous functions (Dragomir et al., 2018). Notably, small RNAs have been found to associate with mitotic chromosomes and play crucial regulatory roles; for instance, small interfering RNAs (siRNA) have been implicated in chromosomes segregation (Huang et al., 2015). These findings make it tempting to speculate that distinct small RNA species are present on mitotic chromosomes and play important roles there. Thus, identification and characterization of mCASRs represent a scientifically valuable endeavor, with the potential to uncover novel components of mitotic chromosomes and novel functions of small RNAs.This study addresses this gap by performing high-throughput sequencing of mCASRs from mouse 3T3 cells under three different salt conditions and comparing them with small RNAs isolated from interphase nuclei. Different types of salt buffers can elute components with varying binding affinities to mitotic chromosomes, while maintaining the stability of the core chromosome structure and morphology (Nishino et al., 2012, Meng et al., 2016). Therefore, we investigated mCASRs under different washing conditions to establish a catalogue of mCASRs that are stably and tightly bound to chromosomes. Overall, for the first time, we provide a dataset cataloguing small RNAs stably bound to mitotic chromosomes in mouse 3T3 cells. The catalogue was generated by stringently aligning and clustering sequencing reads obtained from three independent salt-wash conditions. From an average of >91,000 read clusters, we identified 132 highly and stably associated clusters under all three conditions. Among them, 72 and 6 clusters matched annotated mouse miRNAs and piRNAs, respectively; 6 were likely TSS-associated RNAs; 48 (36.4%) could not be mapped to known small RNAs, of which 13 were predicted as potential novel miRNAs based on structure and sequence features.This dataset constitutes the first comprehensive catalogue of small RNAs tightly associated with mitotic chromosomes in mouse cells, demonstrating that specific small RNAs remain stably bound across distinct experimental conditions. It provides valuable insights into the diversity of chromosomal components and reveals that small RNAs are more widely distributed across subcellular compartments than previously recognized, with notable localization on chromosomes. Our preliminary analysis further transforms the raw data into immediately actionable resource:(1) 132 high-confidence chromosome-bound clusters distilled from three conditions, guiding validation or discovery efforts; (2) annotation of 72 microRNA (miRNAs), 6 putative Piwiinteracting RNA (piRNA), 6 putative TSS-associated (TSSa) RNAs and 13 putative novel miRNAs, providing targets for mechanistic assays. Together, this work lays the foundation for future studies on the functions of small RNAs in chromosomal structuring and mitotic process.An overview of our experimental approach is shown in Figure S1. In short, demecolcine-trapped mitotic cells were isolated and subjected to hypotonic treatment to lyse the plasma membrane. After crude mitotic chromosomes were isolated by low-speed centrifugation, they were treated with different buffers to wash chromosomes. Then, isolate highly pure mitotic chromosomes (containing RNA) by sucrose gradient centrifugation (5%-60%) and collect the 15%-35% fractions. Then, we isolated the RNAs for small RNA library preparation and sequencing. A novel aspect of our protocol lies in the use of different types of buffer washes to isolate the most stably and tightly bound small RNAs associated with mitotic chromosomes.Mouse 3T3 cells (NIH/3T3, ATCC® CRL-1658) were cultured in DMEM (GIBCO, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal calf serum and penicillinstreptomycin (GIBCO, Life Technologies, Carlsbad, CA, USA).Cells were cultured until they reached approximately 80% confluency. The culture medium was then replaced with fresh medium supplemented with demecolcine (D1925, Sigma-Aldrich, USA) at a final concentration of 100 ng/mL. Cells were incubated under these conditions for 12 h and subsequently washed with phosphate-buffered saline (PBS). Mitotic cells were dislodged by gentle shaking, collected, and pelleted by centrifugation at 200 × g for 5 min at 4°C. Mitotic cell purity were quantified by FACS with PI DNA staining. Cells were fixed in 70% ethanol at 4 °C, treated with RNase A (0.2 µg/µL) and Triton X-100 (0.1% w/v), stained with PI (20 µg/mL), and analyzed on a BD LSRFortessa. ModFit software was used for cell-cycle modeling, confirming about 95% mitotic purity(supplemental figure S2).Mitotic chromosome isolation was performed as previously described (Meng et al., 2016). Briefly, collected mitotic cells were incubated in hypotonic solution (75 mM KCl) at room temperature for 30 min, pelleted at 600 × g for 5 min, and resuspended in polyamine (PA) buffer(15 mM Tris-HCl, 0.2 mM spermine, 0.5 mM spermidine, 0.5 mM EGTA, 2 mM EDTA, 80 mM KCl, 20 mM NaCl, 0.1 mM PMSF, 1 mg/ml digitonin). After incubation on ice for 5 min, cells were homogenized using a Dounce homogenizer. Subsequent steps were conducted at 4°C. The lysate was centrifuged at 190 × g for 3 min to yield supernatant S1. The remaining pellet was resuspended, homogenized twice, and centrifuged again to obtain supernatant S2. S1 and S2 were pooled and centrifuged at 420 × g for 5 min to remove debris, followed by centrifugation at 1,750 × g for 10 min.To acquire stably and tightly bound small RNAs, we applied differential buffer washes to remove loosely or nonspecifically associated RNAs, thereby enriching for c

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