Abstract
Formalin-fixed, paraffin-embedded (FFPE) samples represent the gold standard for storage of pathology samples. Here we describe pathology tissue chromatin immunoprecipitation (PAT-ChIP), a technique for extraction and high-throughput analysis, by techniques such as ChIP-seq, of chromatin derived from FFPE samples. Technically, the main challenge of PAT-ChIP is the preparation of good-quality chromatin from FFPE samples. Here we provide a detailed explanation of the methodology used, the choice of reagents and the troubleshooting steps required to establish a robust chromatin preparation procedure. Other steps have also been adapted from existing techniques to optimize their use for PAT-ChIP-seq. The protocol requires 4 d from the start to the end of the PAT-ChIP procedure. PAT-ChIP provides, for the first time, the chance to perform analyses of histone modifications and transcription factor binding on a genome-wide scale using patient-derived FFPE samples. This technique therefore allows the immediate use of pathology archives (even those that are several years old) for epigenetic analyses and the identification of candidate epigenetic biomarkers or targets.
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Acknowledgements
This study was supported by grants from the Italian Association for Cancer Research (AIRC), the European Economic Community (Epitron), the Italian Ministries of Education, Universities and Research (MIUR) and the Ministero della Salute to M.F. and S.M. I.B. is a fellow of the Italian Foundation for Cancer Research (FIRC).
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M.F. and S.M. designed the experiments and wrote the paper; M.F. and S.A. performed the experiments; M.F. and I.B. conducted the bioinformatic analyses.
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Supplementary Fig. 1
Analysis of chromatin integrity following ChIP and PAT-ChIP. (a) Agarose gel electrophoresis analysis of purified DNA from chromatin obtained from leukemic spleen cells treated for classical ChIP (Cells), or for PAT-ChIP (FFPE). (b) Cells-ChIP-Seq and PAT-ChIP-Seq of the H3K4Me3 histone mark, and analysis of peak distribution with respect to the following genomic regions: TSS (±2.5kb) of RefSeq annotated genes; intergenic, intron and exon regions. (TIFF 6376 kb)
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Fanelli, M., Amatori, S., Barozzi, I. et al. Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue. Nat Protoc 6, 1905–1919 (2011). https://doi.org/10.1038/nprot.2011.406
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DOI: https://doi.org/10.1038/nprot.2011.406
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