FFPE‐ATAC: A Highly Sensitive Method for Profiling Chromatin Accessibility in Formalin‐Fixed Paraffin‐Embedded Samples

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

Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations

“…The ability to study molecular changes from large patient cohorts within the same pathology and correlate results with the clinical signs of that specific moment is a game changer in biomarker discovery. Many advanced techniques nowadays are optimized for archived specimens, 5 , 6 , 7 , 8 , 27 , 28 nonetheless due to their user-friendliness and cost-effectiveness, RT-qPCR-based approaches are the most widely used. RT-qPCR-based microfluidic cards are user-friendly nanoliter-scaled techniques that enable the detection of hundreds of miRNAs simultaneously, starting from as little as 1 pg of total RNA.…”

“…38 Archived specimens represent valuable resources in clinical research, which is proven by the fact that novel molecular techniques are optimized considering the limitations of this type of sample. 5 , 7 , 27 , 28 , 39 RNA in fixed and archived samples is usually highly fragmented requiring preliminary RNA quality and integrity analysis before performing expensive and highly sensitive experiments. 27 , 40 However, previously published experiments highlighted that miRNAs are usually not affected by fragmentation, being themselves around 20 nucleotide-long fragments.…”

“… 1 , 2 , 3 , 4 This research field is particularly advanced in cancer and is associated with access to fresh surgical tissues required for molecular profiling, while in a plethora of clinical fields, neurology included, specific studies need to be designed and specimens taken exclusively for study purposes. However, efforts have been made to assess archived specimens, taken for diagnostic purposes, and their quality and utility for molecular profiling, 5 , 6 , 7 , 8 which paved new avenues for molecular-based retrospective studies on archived specimens taken for diagnostic purposes or in clinical trials.…”

Emerging Approaches to Profile Accessible Chromatin from Formalin-Fixed Paraffin-Embedded Sections

“…Beyond the application of ATAC to interrogate model organism and cell line epigenomes, recent efforts have explored if the enzyme could be applied to clinical studies on FFPE tissue sections [ 71 , 72 , 73 , 74 , 75 ]. Obtaining PCR amplicons from Tn5-based approaches requires two independent tagmentation events in opposing orientation and in close proximity (<~700 bp).…”

“…This inherently reduces library complexity and effective yields and is exacerbated by the highly damaged DNA in FFPE material. To address this, a recent approach followed Tn5 tagmentation with an in vitro transcription (IVT) step, such that a single insertion event could be amplified by T7 RNA polymerase [ 72 , 73 , 74 , 75 ]. While standard ATAC-seq yielded some success in nuclei isolated from mouse FFPE liver and kidney, the Tn5-IVT-modified approach improved the library complexity, signal-to-noise ratio, and other key metrics.…”