Mapping the chromatin landscape of the mouse immune system with low-input automated CUT&RUN 2260000

Abstract Introduction Understanding how immune cells develop and function requires insight into the epigenomic mechanisms that regulate gene expression. While many genomic studies focus on transcriptional outputs, changes in the chromatin landscape play a central role in shaping lineage commitment. The mammalian immune system is composed of highly diverse and dynamic cell types, but detailed epigenomic studies have been severely limited by technical challenges in profiling rare cell populations. Methods We developed and validated a low-input, automated CUT&RUN workflow that incorporates standardized sample preparation to ensure reliable generation of data at the consortium scale. This method minimizes sample handling and applies internal controls to monitor assay performance during experimental and sequencing stages. Results Extensive optimization of assay conditions and antibody reagents enabled robust mapping of histone post-translational modifications (PTMs) from as few as 10,000 cells per reaction. Applying this approach, we profiled >170 immune subpopulations collected from 11 ImmGen consortium labs over two years. Conclusion These innovations establish a scalable, high-resolution platform for profiling chromatin landscapes from minimal cell inputs. Our automated CUT&RUN pipeline enables standardized, reproducible analysis across diverse immune cell types and can distinguish technical issues from true biological insights. Together, these advances lay the foundation for a companion study presenting the first comprehensive epigenomic atlas of immune lineages and provide a framework for studying chromatin regulation in rare or limited samples across the life sciences. Funding Source NIH R44 AI167215 Topic Categories Technological Innovations in Immunology (TECH)