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Microbiology JoVE (Open Access) Citable · DOI

Chromatin Isolation by RNA Purification (ChIRP)

DOI: 10.3791/3912-v
What you'll learn
  • ✓ Perform chromatin cross-linking and cell lysis to preserve RNA-chromatin interactions
  • ✓ Apply sonication to shear DNA for chromatin fragmentation
  • ✓ Execute ChIRP pulldown using anti-sense oligonucleotide probes to isolate lncRNA-bound genomic sites
  • ✓ Analyze enrichment of target lncRNAs by quantitative methods
Protocol

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Difficulty
advanced
Total time
~4–6 hours per sample (cross-linking through ChIRP isolation)
Model organism
HEK293
Biosafety
BSL-1

Steps

1
Cross-link cells to preserve RNA-chromatin interactions

Treat cultured cells with formaldehyde to covalently cross-link RNA to chromatin, stabilizing lncRNA-genomic binding sites for downstream analysis.

▶ 01:25
2
Lyse cells and isolate chromatin-bound material

Permeabilize and lyse cross-linked cells under controlled conditions to release chromatin complexes while maintaining RNA-DNA associations.

▶ 02:58
3
Shear DNA by sonication into manageable fragments

Apply ultrasonic waves to fragment cross-linked chromatin into small, workable pieces suitable for pulldown enrichment.

▶ 03:37
4
Perform chromatin isolation by RNA purification

Hybridize anti-sense tiling oligonucleotide probes to target lncRNAs and capture probe-bound chromatin complexes using streptavidin beads.

▶ 05:13
5
Analyze enrichment of target long noncoding RNAs

Quantify recovered lncRNAs and associated genomic sequences by qPCR or sequencing to validate ChIRP specificity and identify bound genomic sites.

▶ 08:43
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