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FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues [mouse]

ABSTRACT: Fixed-Tissue ChIP-seq for H3K27Ac profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in FFPE tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27Ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65°C in more stringent detergent conditions and a minimized sonication step, to produce robust genome-wide H3K27Ac maps from clinical samples. We demonstrate FiTAc-seq suitability to generate high quality enhancer landscapes and SE annotation in a number of archived FFPE samples from distinct tumor types. This approach will be of great interest both for basic and clinical researchers. The entire protocol from FFPE blocks to sequence ready library can be accomplished within 4 days.

ORGANISM(S): Mus musculus

PROVIDER: GSE140805 | GEO | 2020/06/26

REPOSITORIES: GEO

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FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues [human]

Project description:Fixed-Tissue ChIP-seq for H3K27Ac profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in FFPE tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27Ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65°C in more stringent detergent conditions and a minimized sonication step, to produce robust genome-wide H3K27Ac maps from clinical samples. We demonstrate FiTAc-seq suitability to generate high quality enhancer landscapes and SE annotation in a number of archived FFPE samples from distinct tumor types. This approach will be of great interest both for basic and clinical researchers. The entire protocol from FFPE blocks to sequence ready library can be accomplished within 4 days.

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FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues [human PNET]

Project description:Fixed-Tissue ChIP-seq for H3K27Ac profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in FFPE tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27Ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65°C in more stringent detergent conditions and a minimized sonication step, to produce robust genome wide H3K27Ac maps from clinical samples. We demonstrate FiTAc-seq suitability to generate high quality enhancer landscapes and SE annotation in a number of archived FFPE samples from distinct tumor types. This approach will be of great interest both for basic and clinical researchers. The entire protocol from FFPE blocks to sequence ready library can be accomplished within 4 days.

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FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues

Project description:FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues

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FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues [mouse]

Project description:FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues [mouse]

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FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues [human]

Project description:FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues [human]

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Optimizing microarray gene expression profiling workflow for formalin-fixed paraffin-embedded tissues

Project description:The use of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues for high-throughput molecular techniques, such as microarray gene expression profiling has become widespread in molecular research area. However, working with FFPE tissues is challenging because of degradation, cross-linking with proteins, and RNA chemical modifications. Also, there is no generally accepted procedure for RNA extraction to microarray analysis. Thus, there is a need for a standardized workflow for FFPE samples to study microarray transcriptome profiling. Therefore, the main purpose of this study was to conduct a standardized process from deparaffinization to RNA extraction and microarray gene expression analysis. Firstly, deparaffinization procedure was optimized for FFPE samples and then Trizol, PicoPure RNA isolation kit, and Qiagen RNeasy FFPE kit performances were compared in terms of yield and purity. Finally, two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were also evaluated to determine which combination gives the best percentage of present call. Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol and U133_X3P array combination gave a significantly higher percentage of present calls than the 3’ IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms. These results present a workflow for microarray gene expression profiling of FFPE tissues. The findings also indicate that sufficient quality gene expression data can be obtained from FFPE-derived RNA.

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FiTAc-seq: Fixed-Tissue ChIP-seq for H3K27Ac profiling and super-enhancer analysis on FFPE tissues [human PNET]

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Project description:FFPE tissue samples have long been considered challenging for sc-/sn-RNA sequencing assays. In this study, we developed a novel snRNA-seq workflow, snPATHO-seq, for FFPE tissue samples utilising isolated high-quality nuclei from FFPE samples and 10X Genomics FLEX chemistry. The snPATHO-seq demonstrated consistent performance with the established 10X Genomics 3' assay. In addition, it allows more accurate single-nucleus and spatial transcriptomics integration with the FFPE Visium dataset generated from the same FFPE block. We envision this new protocol can facilitate the transcriptomic interpretation of millions of FFPE archives generated and simplify the sample collection process and data generation.

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snPATHO-seq: unlocking the FFPE archives for single nucleus RNA profiling [snRNA-seq]

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Automated array-CGH optimized for archival formalin-fixed, paraffin-embedded tumor material

Project description:BACKGROUND: Array Comparative Genomic Hybridization (aCGH) is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE) tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH. METHODS: In this report, we first used DNA from a cancer cell-line (SKBR3) to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use. RESULTS: Our protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method. CONCLUSION: This report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility.

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