Enhanced PAT-ChIP (EPAT-ChIP), a new procedure for reliable genome-wide studies in clinical FFPE tissues
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ABSTRACT:
PROVIDER: PRJNA483066 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7609365.fastq.gz | Fastqsanger.gz | |||
| SRR7609366.fastq.gz | Fastqsanger.gz | |||
| SRR7609367.fastq.gz | Fastqsanger.gz | |||
| SRR7609368.fastq.gz | Fastqsanger.gz | |||
| SRR8098973.fastq.gz | Fastqsanger.gz |
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Project description:Chromatin immunoprecipitation (ChIP) has allowed the study of protein-DNA interactions in chromatin, bringing new insights into the role of histone post-translational modifications (HPTMs) in cancer and other diseases. The coupling of ChIP with next-generation sequencing (NGS) techniques has revealed the distribution of HPTMs over the entire genome allowing the identification of altered epigenetic profiles in pathological conditions. However, until recently, these studies have been limited to cultured cells or fresh/frozen tissues. The development of a new technique named PAT-ChIP has enabled chromatin studies in formalin-fixed paraffin-embedded (FFPE) tissues, opening the doors to a huge number of samples stored in pathology archives and related clinical information. However, chromatin studies in FFPE archival samples can be hindered by the extensive formalin fixation to which these tissues are routinely subjected. Here, we describe enhanced PAT-ChIP (EPAT-ChIP), a new ChIP procedure that facilitates epigenomic studies in archival FFPE tissues. EPAT-ChIP improves the extraction of soluble chromatin, allowing the study of multiple histone marks from limited amounts of starting material. In addition, EPAT-ChIP assures higher yields of final immunoselected DNA, especially when poorly represented histone marks (e.g. narrow peaks of H3K4me3) are investigated, and the consequent production of reliable NGS libraries. In this procedure, FFPE samples are subjected to heat-mediated limited reversal of crosslinking (LRC) which predisposes the tissue to better release soluble chromatin after sonication without altering the antigenic potential of its biomolecules. After investigating differentially-fixed normal colon specimens specifically produced for this study, ePAT-ChIP was successfully used to study three different HPTMs (H3K4me3, K3K27me3, H3K27ac) at genome-wide level in an archival invasive breast carcinoma (IBC) sample.
2018-11-20 | GSE117752 | GEO
Project description:Epigenetic alterations in the pattern of DNA and histone modifications play a crucial role in cancer development. Analysis of patient samples, however, is hampered by technical limitations in the study of chromatin structure from pathology archives that typically consist of heavily fixed, paraffin-embedded material. ChIP assays (with anti-H3K4me3 and anti-H3K27me3 antibodies) were performed by using formaldehyde-fixed paraffin-embedded (FFPE) mouse spleen sections (half portion of the spleen) and comparing it to the canonical ChIP procedure applied by using isolated cells from the other portion of the same spleen.
2010-12-02 | E-GEOD-21449 | biostudies-arrayexpress
Project description:BackgroundThe introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation (ChIP) from formalin-fixed paraffin-embedded (FFPE) tissues, has extended the application of chromatin studies to clinical patient samples. However, extensive crosslinking introduced during routine tissue fixation of clinical specimens may hamper the application of PAT-ChIP to genome-wide studies (PAT-ChIP-Seq) from archived tissue samples. The reduced efficiency in chromatin extraction from over-fixed formalin archival samples is the main hurdle to overcome, especially when low abundant epigenetic marks (e.g., H3K4me3) are investigated.ResultsWe evaluated different modifications of the original PAT-ChIP protocol to improve chromatin isolation from FFPE tissues. With this aim, we first made extensive usage of a normal human colon specimen fixed at controlled conditions (24 h, 48 h, and 72 h) to mimic the variability of tissue fixation that is most frequently found in archived samples. Different conditions of chromatin extraction were tested applying either diverse sonication protocols or heat-mediated limited reversal of crosslinking (LRC). We found that, if compared with canonical PAT-ChIP protocol, LRC strongly increases chromatin extraction efficiency, especially when 72-h fixed FFPE samples are used. The new procedure, that we named enhanced PAT-ChIP (EPAT-ChIP), was then applied at genome-wide level using an archival sample of invasive breast carcinoma to investigate H3K4me3, a lowly abundant histone modification, and H3K27me3 and H3K27ac, two additional well-known histone marks.ConclusionsEPAT-ChIP procedure improves the efficiency of chromatin isolation from FFPE samples allowing the study of long time-fixed specimens (72 h), as well as the investigation of low distributed epigenetic marks (e.g., H3K4me3) and the analysis of multiple histone marks from low amounts of starting material. We believe that EPAT-ChIP will facilitate the application of chromatin studies to archived pathology samples, thus contributing to extend the current understanding of cancer epigenomes and enabling the identification of clinically useful tumor biomarkers.
| S-EPMC6240272 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below. The reliability of differential expression analysis on FFPE expression profiles from Affymetrix arrays is questionable, due to the wide range of percent-present values reported in studies which profiled FFPE samples on Affymetrix arrays. Moreover the validity of externally defined gene-modules in FFPE microarray expression profiles is unknown. Using eight breast cancer tumors with available frozen and FFPE samples, five sample-matched data sets were generated from different combination of Affymetrix arrays, amplification-and-labeling kit and sample preservation method. The reliability of differential expression analysis was investigated by developing de novo ER/HER2 pathway gene-modules from matched data sets and validating it on external data set using ROC analysis. Spearman's rank correlation coefficient of module scores between matched FFPE-frozen expression profiles was used to measure reliability of externally defined gene-modules in FFPE expression profiles. Independent of array/amplification-kit/sample preservation method used, de novo ER/HER2 gene-modules derived from all matching data sets showed similar prediction performance during independent validation (AUC range; ER: 0.92-0.95, HER2: 0.88-0.91), except for de novo HER2 gene-module derived from FFPE data set with 3'IVT kit (AUC: 0.67-0.72). Further not all gene-module based biological signals present in frozen expression profiles can be recovered from matching FFPE microarray expression profiles using the currently available FFPE specific sample preparation kits. The gene-module based biological signal extracted from FFPE RNA, using microarrays, may not be as reliable as that from their frozen counterpart, if the sample preparation protocol used with FFPE RNA failed to recover relevant genes involved in the biological signal.
2018-09-05 | GSE93338 | GEO
Project description:Setaria viridis strain:PAT-5 | cultivar:PAT-5 Genome sequencing
| PRJNA334531 | ENA
Project description:Feasibility of Developing Reliable Gene Expression Modules from FFPE Derived RNA Profiled on Affymetrix Arrays
| PRJNA360624 | ENA
Project description:Coronary artery disease (CAD) poses a worldwide health threat. Compelling evidence shows that pericardial adipose tissue (PAT), a brown-like adipose adjacent to the external surface of the pericardium, is associated with CAD. However, the specific molecular mechanisms of PAT in CAD are elusive. For characterizing human PAT and explore its association with CAD, the transcriptome characteristics were assessed in 5 CAD patients and 4 controls via RNA-sequencing.
2021-07-30 | GSE179397 | GEO
Project description:Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin.
2019-03-18 | GSE128202 | GEO
Project description:The Arabidopsis genome encodes 3 PAT mRNA decapping factors: PAT1(AT1g79090), PATH1 (AT3g22270) and PATH2 (AT4g14990). We generated single, double and triple knockouts (KOs) of the three PATs by CRISPR/CAS9-mediated genome editing in the summ2-8 background to avoid immune activation. The 6 weeks-old soil-grown pat triple mutants exhibited markedly stunted growth compared to the other pat single or double mutants indicating functional redundancy of PATs in regulating plant development. To identify genes which affect different developmental programs regulated by PATs, we performed RNA-seq from plants of pat1-1path1-4path2-1summ2-8 and all single and double mutant combinations.
2024-01-26 | GSE253757 | GEO
Project description:Optimized ChIP-seq procedure facilitates hormone receptor profiling in human tumors
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