A Highly Sensitive Method to Efficiently Profile the Histone Modifications of FFPE Samples

Zhao, Linxuan; Polavarapu, Vamsi Krishna; Yadav, Ram Prakash; Xing, Pengwei; Chen, Xingqi

doi:10.21769/bioprotoc.4418

Cited by 4 publications

(7 citation statements)

References 22 publications

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“…no. BP531) were activated just before use with Ca ++ and Mn ++ as described 21 . Frozen whole-cell aliquots were thawed at room temperature, split into PCR tubes and 5 µL ConA beads were added with gentle vortexing.…”

Section: Methodsmentioning

confidence: 99%

“…Modifications to the standard ATAC-seq protocol were required to make it suitable for FFPEs, including nuclei isolation following enzymatic tissue disruption and in vitro transcription with T7 RNA polymerase 18 , 19 . The same group also similarly modified CUT&Tag and included an epitope retrieval step using ionic detergents and elevated temperatures, which they termed F FPE tissue with A ntibody-guided C hromatin T agmentation with seq uencing (FACT-seq) 20 , 21 . However, FACT-seq is a 5-day protocol even before sequencing, and the many extra steps required relative to CUT&Tag have raised concerns about experimental variability 4 .…”

Section: Introductionmentioning

confidence: 99%

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Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag

Henikoff,

Ahmad

et al. 2023

Nat Commun

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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 CUTAC protocol either in single tubes or directly on slides produce high-resolution maps of paused RNA Polymerase II at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumors and identify and map regulatory element markers with high confidence and precision, including microRNAs not detectable by RNA-seq. Our simple workflows make possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag

Henikoff,

Ahmad

et al. 2023

Nat Commun

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“…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).…”

Section: Genome-wide Profiling Of Open Chromatinmentioning

confidence: 99%

“…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.…”

Section: Genome-wide Profiling Of Open Chromatinmentioning

confidence: 99%

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

Sunitha Kumary,

Venters,

Raman

et al. 2024

Epigenomes

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Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of ‘open’ chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.

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“…Beyond the application of ATAC to interrogate the epigenomes of model organisms and cell lines, recent efforts have sought to enable clicinal studies from FFPE tissue sections [67][68][69][70][71] . To prepare amplicons from Tn5-based approaches, two independent tagmentation events are required in opposing orientation and in close proximity (<~700 bp).…”

Section: Tn5 Transposon Tagmentation Of Accessible Chromatin (Atac-seq)mentioning

confidence: 99%

Emerging Approaches to Profile Accessible Chromatin from Ffpe Sections

Sunita Kumary,

Venters,

Raman

et al. 2024

Preprint

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Nucleosomes are non-uniformly distributed across eukarytic genomes, with stretches of ‘open’ chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and damaged genomic material. Here we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.

show abstract

A Highly Sensitive Method to Efficiently Profile the Histone Modifications of FFPE Samples

Cited by 4 publications

References 22 publications

Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag

Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag

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

Emerging Approaches to Profile Accessible Chromatin from Ffpe Sections

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