Biology subjects

Thekkedam, C.

Publications and source records attributed to Thekkedam, C..

3 recordsLinked to original sources

bioRxiv2026

A scalable MNase-seq framework for reproducible nucleosome profiling across pluripotent stem cell and cardiomyocyte models

Micrococcal nuclease (MNase) digestion is widely used to profile chromatin accessibility and nucleosome footprinting. However, its application is often limited by sensitivity to reaction conditions, high cell input requirements, and the lack of standardized protocols across cell types. Here we developed a robust MNase workflow encompassing buffer composition, DNA purification chemistry, fixation and decrosslinking parameters, cell input scalability, and an in-house yeast spike-in for quantitative normalization. We validated this unified framework across human induced pluripotent stem cells (hiPSCs), hiPSC-derived cardiomyocytes at multiple differentiation stages, primary murine embryonic cardiac cells, and adult mouse cardiomyocytes, and demonstrated comparable digestion efficiencies and kinetics despite marked differences in cellular architecture and chromatin organization. Genome-wide MNase-seq in hiPSCs, combined with the nucMACC bioinformatic pipeline, resolved concentration-dependent nucleosomal occupancy and precise nucleosome positioning at pluripotency-related regulatory elements. This modular, end-to-end, and scalable workflow provides a standardized platform for reproducible MNase-based chromatin profiling across diverse in vitro and in vivo models. TEASERA unified, rapid, and scalable MNase-seq workflow for reproducible mononucleosomal and subnucleosomal profiling from stem cells to adult cardiomyocytes. HIGHLIGHTSO_LISystematic MNase optimization across buffer, DNA purification, and cell input variables C_LIO_LIUnified workflow validated in hiPSCs, hiPSC-CMs, embryonic, and adult cardiomyocytes C_LIO_LIFixed-cell protocol enables weeks of storage without loss of DNA quality C_LIO_LICost-effective yeast spike-in ensures quantitative normalization for MNase-seq C_LIO_LIGenome-wide analyses confirm robust and precise nucleosome positioning at regulatory elements C_LI

genomics↗

bioRxiv2026

Proteo-transcriptomics and morphometrics of teleost cardiac cells define regulatory networks and exercise-induced cardiomyocyte hypertrophy and hyperplasia

Zebrafish and medaka are powerful cardiovascular models, yet cellular and molecular investigations of adult heart cells have been constrained by suboptimal dissociation and characterization methods. To overcome these barriers, we developed a physiological-temperature workflow that generates high-yield, viable single-cell suspensions for FACS, imaging, and low-input molecular profiling. Using transgenic fluorescent reporters, we consistently isolate [~]6,000 cardiomyocytes per adult zebrafish ventricle and [~]12,000 from medaka, preserving cellular, structural, and molecular integrity. Single-cell morphometrics revealed cardiomyocyte heterogeneity and demonstrated that swimming exercise induces both hypertrophy and hyperplasia, while ventricular injury triggers expansion of regenerative gata4 cardiomyocytes. We integrated proteomics and RNA-seq from FACS-purified cells to construct cell-type-specific proteo-transcriptomic atlases. Functional enrichment, transcription factor, and network analyses identified protein hubs and regulatory circuits defining cardiomyocyte and endothelial cell identity. Our platform delivers cell-type-resolved molecular datasets of adult teleost cardiac cells, establishing a systems-level resource for heart regeneration research, cardiovascular disease modeling, and drug discovery. TeaserA robust workflow for isolation, FACS, imaging, and multi-omics profiling of cardiomyocytes and cardiac endothelial cells in fish. HIGHLIGHTSO_LITemperature-optimized dissociation and standardized FACS enable high-yield isolation of viable cardiac cells. C_LIO_LISingle-cell imaging uncovers morphological heterogeneity in adult ventricular cardiomyocytes. C_LIO_LISustained exercise induces both hypertrophy and hyperplasia of zebrafish cardiomyocytes. C_LIO_LIProteo-transcriptomics defines core molecular programs and interaction networks in cardiomyocytes and endothelial cells. C_LI

systems biology↗

bioRxiv2025

OpenEMMU: a versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

5-Ethynyl-2-deoxyuridine (EdU) has transformed DNA replication and cell cycle analyses through fast and efficient click chemistry detection. However, commercial EdU kits are expensive, contain proprietary reagents, are suboptimal for antibody multiplexing, and are challenging to use with larger biological specimens, highlighting the need for innovation. Here, we report Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics (OpenEMMU), an optimized, affordable, and user-friendly click chemistry resource using off-the-shelf reagents. OpenEMMU improves the efficiency, brightness, and multiplexing capabilities of EdU thymidine analog staining with both non-conjugated and conjugated antibodies across various cell types. We validated its effectiveness for fluorescent imaging of nascent DNA in developing embryos and organs, including the embryonic heart and forelimbs, and in 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled 3D imaging of DNA synthesis in zebrafish larvae at fine resolution. This opens new avenues for insights into organismal development and growth, cell proliferation, and DNA replication, with precision and flexibility that surpass traditional methods. TeaserAn open-source and cost-effective resource for DNA replication profiling and cell cycle analysis using click chemistry.

developmental biology↗