✓Identify proteins and RNAs associated with specific genomic regions
✓Analyze immunoprecipitated chromatin by qPCR and sequencing methods
Protocol
Biopharma Insights The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.
Difficulty
advanced
Total time
~2–3 days (including cell culture preparation, crosslinking, immunoprecipitation, and analysis)
Biosafety
BSL-1
Steps
1
Prepare tagged chromatin for immunoprecipitation
Engineer cells to express DNA-binding molecules tagged with affinity handles, then prepare chromatin from cultured cells through crosslinking and sonication to achieve the desired fragment size.
▶ 00:41
2
Perform chromatin immunoprecipitation with enChIP
Use affinity-tagged DNA-binding molecules to selectively pull down specific genomic regions and their associated complexes from sonicated chromatin under immunoprecipitation conditions.
▶ 04:16
3
Analyze immunoprecipitated chromatin composition
Extract DNA and RNA from immunoprecipitated samples, then quantify enrichment at target genomic regions and identify associated molecules by qPCR, sequencing, or mass spectrometry.
▶ 06:41
4
Interpret enChIP results from telomeres and promoters
Examine representative results showing enChIP applications to telomeric regions and the IRF-1 promoter, demonstrating recovery of associated regulatory proteins and RNAs.
▶ 08:08
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