High-Efficiency Genomic Mapping of Chromatin-Associated Targets with CUT&RUN.

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

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. 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. 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. 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. NIH R44 AI167215 Technological Innovations in Immunology (TECH)

CUT&Tag for Chromatin Profiling in Cancer Tissue.

Defining the levels of the chromatin landscape using the I-CATCH system

Alterations of the epigenome, including histone tail post-translational modifications (PTMs), have been shown to regulate the expression of genes important for driving multiple cellular processes including cellular differentiation, aging and tumorigenesis. Although the ability to measure histone tail PTM levels is integral to the understanding of chromatin biology, because of technological limitations, the proportion of nucleosomes that have histone tail PTMs at genomic loci remain undefined. To overcome this limitation, we developed the i mmuno-profiling of d Ca s9 t argeted ch romatin (I-CATCH) system, a technology designed to measure and normalize the levels of nucleosomes and co-occurring histone tail PTMs at targeted loci. Here, we show that the I-CATCH system has high analytical performance for measuring the levels of histone tail PTMs and we report the normalized levels of multiple histone tail PTMs at different genomic loci from both HeLa cells and human plasma. While further studies are required to determine the full utility of the I-CATCH system, we are hopeful that the I-CATCH system will help enable the ability to investigate new important features of chromatin biology.

Genomic Profiling of Chromatin State Using CUT&Tag.

Chromatin Immunoprecipitation Sequencing in Cancer Cells.

The protocols for ChIP-seq are outlined, including immunoprecipitation, library preparation for high-throughput sequencing, and bioinformatics analysis of sequencing data that the authors routinely perform in their lab groups, and include troubleshooting tips for each step.

Protocol for high-resolution mapping of RNA-associated chromatin using chromatin isolation by RNA purification-tag

Summary Mapping the genomic binding sites of long non-coding RNAs (lncRNAs) is essential for understanding their regulatory functions. Here, we present a protocol for integrating RNA hybridization with in situ tagmentation using chromatin isolation by RNA purification-tag (ChIRP-Tag). We describe steps for performing cell permeabilization, hybridization of biotinylated probes to target RNAs, and recruiting Protein A/G-Tn5 transposase via antibodies to catalyze in situ library construction. This protocol captures RNA-associated chromatin with high sensitivity and spatial resolution across various cell types. For complete details on the use and execution of this protocol, please refer to Fu et al.1