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  • Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue.

Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue.

Nature protocols (2011-11-16)
Mirco Fanelli, Stefano Amatori, Iros Barozzi, Saverio Minucci
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.

MATERIALS
Product Number
Brand
Product Description

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Anti-acetyl-Histone H4 Antibody, serum, Upstate®
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Anti-CTCF Antibody, serum, Upstate®
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Anti-acetyl-Histone H3 Antibody, from rabbit
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Anti-trimethyl-Histone H3 (Lys27) Antibody, Upstate®, from rabbit
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DNA Molecular Weight Marker XIV (100 bp ladder), solution, pkg of 50 μg (in 200 μl)