Kugel, S. | Search bioRxiv

bioRxiv2023

Epigenomic analysis of Formalin-Fixed Paraffin-Embedded samples by CUT&Tag

For more than a century, Formalin Fixed Paraffin Embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our single-tube CUTAC protocol are sufficient to produce high-resolution maps of paused RNA Polymerase II (RNAPII) at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumor specimens and identify regulatory element markers with high confidence and precision. Our simple work-flow is suitable for automation, making possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.

cancer biology↗

bioRxiv2023

Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ.

Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localized in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNA Xist as models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization of Xist and other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.

molecular biology↗