FFPE‐ATAC: A Highly Sensitive Method for Profiling Chromatin Accessibility in Formalin‐Fixed Paraffin‐Embedded Samples. Issue 8 (22nd August 2022)

• Record Type: Journal Article
• Authors: Yadav, Ram Prakash; Polavarapu, Vamsi Krishna; Xing, Pengwei; Chen, Xingqi
• Is Part Of: Current protocols. Volume 2:Issue 8(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-08-22
• Subjects: chromatin accessibility -- formalin‐fixed paraffin‐embedded (FFPE) sample -- in vitro transcription -- T7‐Tn5‐mediated transposition; Life sciences -- Laboratory manuals -- Periodicals; Biology -- Laboratory manuals -- Periodicals; Life sciences -- Technique -- Periodicals; Biology -- Technique -- Periodicals; 570.028
• DOI: 10.1002/cpz1.535
• Languages: English
• ISSNs: 2691-1299

Abstract

Abstract: In basic and translational cancer research, the majority of biopsies are stored in formalin‐fixed paraffin‐embedded (FFPE) samples. Chromatin accessibility reflects the degree to which nuclear macromolecules can physically interact with chromatinized DNA and plays a key role in gene regulation in different physiological conditions. As such, the profiling of chromatin accessibility in archived FFPE tissue can be critical to understanding gene regulation in health and disease. Due to the high degree of DNA damage in FFPE samples, accurate mapping of chromatin accessibility in these specimens is extremely difficult. To address this issue, we recently established FFPE‐ATAC, a highly sensitive method based on T7‐Tn5‐mediated transposition followed by in vitro transcription (IVT), to generate high‐quality chromatin accessibility profiles with 500–50, 000 nuclei from a single FFPE tissue section. In FFPE‐ATAC, which we describe here, the T7‐Tn5 adaptors are inserted into the genome after FFPE sample preparation and are unlikely to sustain the DNA breakage that occurs during reverse cross‐linking of these samples. It should, therefore, remain at the ends of broken accessible chromatin sites after reverse cross‐linking. IVT is then used to convert the two ends of the broken DNA fragments to RNA molecules before making sequencing libraries from the IVT RNAs and further decoding Tn5 adaptor insertion sites in the genome. Through this strategy, users can decode the flanking sequences of the accessible chromatin even if there are breaks between adjacent pairs of T7‐T5 adaptor insertion sites. This method is applicable to dissecting chromatin profiles of a small section of the tissue sample, characterizing stage and region‐specific gene regulation and disease‐associated chromatin regulation in FFPE tissues. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Nuclei isolation from FFPE tissue samples Basic Protocol 2 : T7‐Tn5 transposase tagmentation, reverse‐crosslinking, and in vitro transcription Basic Protocol 3 : Preparation of libraries for high‐throughput sequencing