10.6084/m9.figshare.9976595.v1 Alžběta Němečková Alžběta Němečková Christina Wäsch Christina Wäsch Veit Schubert Veit Schubert Takayoshi Ishii Takayoshi Ishii Eva Hřibová Eva Hřibová Andreas Houben Andreas Houben Supplementary Material for: CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication Karger Publishers 2019 C RISPR/Cas9 DNA replication FISH Immunostaining RGEN-ISL Super-resolution microscopy 2019-10-14 12:42:59 Figure https://karger.figshare.com/articles/figure/Supplementary_Material_for_CRISPR_Cas9-Based_RGEN-ISL_Allows_the_Simultaneous_and_Specific_Visualization_of_Proteins_DNA_Repeats_and_Sites_of_DNA_Replication/9976595 Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)<sub>n</sub> and <i>Arabidopsis</i>-type (TTTAGGG)<sub>n</sub> telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.