The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Summary

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

Abstract

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Introduction

Chromatin immunoprecipitation (ChIP) is a powerful method to study mechanisms of gene regulation by selectively enriching for DNA fragments that interact with a given protein in living cells. Detection methods of ChIP-enriched DNA fragments have evolved as technology improves, from detection of a single locus (standard ChIP-qPCR) to hybridization on oligonucleotide microarrays (ChIP-chip) to high-throughput sequencing (ChIP-seq) ¹. Although ChIP-seq has seen widespread application, chromatin heterogeneity and nonspecific DNA interactions have hampered data quality leading to false positives and imprecise mapping. To circumvent these limitations, Dr. Frank P....

Protocol

Note: Double distilled H₂O or molecular grade equivalent is recommended for all buffers and reactions mixes.

Day 0: Material Preparation and Cell Harvest

1. Buffer Preparation

1. Prepare Lysis Buffers 1 - 3 (Tables 1 - 3) and add 100 µl complete protease inhibitor stock (CPI) to 50 ml of each buffer just prior to use. Prepare CPI stock by dissolving one tablet in 1 ml molecular grade H₂O.
2. Prepare ChIP Buffers (Tables 4 - 7). Add 100 µl CPI stock to 50 ml of Blocking and RIPA buffers. Do not add CPI to Tris and ChIP Elution buffers.....

Results

The following figures illustrate representative results from the ChIP-exo protocol presented here. In contrast to traditional ChIP-seq methodologies with few enzymatic steps, ChIP-exo requires eleven sequentially dependent enzymatic reactions (Figure 1). Thus, care must be taken at each step to ensure that each reaction component is added to its respective reaction master mix. We recommend generating a formulaic spreadsheet based on the reaction Tables to automatically pe.......

Discussion

We present a functional genomic protocol to determine the precise binding location for chromatin interacting proteins in an unbiased, genome-wide manner at near base pair resolution. The most critical step to achieve near base pair mapping resolution is the exonuclease treatment of the ChIP-enriched DNA while the immunoprecipitate remains on the magnetic resin. Ostensibly, protein complexes could potentially block in vivo footprinting of any given subunit (e.g., chromatin remodeling complexes or the nuc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
37% formaldehyde, methanol free, 10 x 10 ml ampules	ThermoFisher Scientific	28908	Section 3
Complete Protease Inhibitor cocktail (CPI)	Roche Life Science	11873580001	Sections 4, 8
Bioruptor sonicator	Diagenode	UCD200	Section 5
15 ml polystyrene tubes	BD Falcon	352095	Section 5
MagSepharose Protein G Xtra beads	GE Healthcare	28-9670-66	Section 6
DynaMag-1.5 ml Side Magnetic Rack	Invitrogen	12321D	Sections 6-17, 22
Mini-Tube Rotator	Fisher Scientific	05-450-127	Sections 7, 22
T4 DNA Polymerase	New England BioLabs	M0203	Section 9
DNA Polymerase I, Klenow	New England BioLabs	M0210	Section 9
10x NEBuffer 2 (10x Reaction Buffer 2)	New England BioLabs	B7002	Sections 9-11, 13, 15, 20
Thermomixer C	Eppendorf	5382	Sections 9-16
ATP	Roche Life Science	010419979001	Sections 9, 11, 13
dNTPs	New England BioLabs	N0447	Sections 9, 12, 19, 23
T4 Polynucleotide Kinase	New England BioLabs	M0201	Sections 9, 13
dATP	New England BioLabs	N0440	Sections 10, 20
Klenow 3'-5' Exo Minus	New England BioLabs	M0212	Sections 10, 20
T4 DNA ligase	New England BioLabs	M0202	Sections 11, 21
Φ-29 DNA Polymerase	New England BioLabs	M0269	Sections 12, 19
10x Φ-29 Buffer	New England BioLabs	B0269	Sections 12, 19
Lambda Exonuclease	New England BioLabs	M0262	Section 14
10x Lambda Buffer	New England BioLabs	B0262	Section 14
RecJf Exonuclease	New England BioLabs	M0264	Section 15
Proteinase K	Roche Life Science	03115828001	Section 16
Glycogen	Roche Life Science	010901393001	Section 18
10x T4 Ligase Buffer	New England BioLabs	B0202	Section 21
AMPure XP (clean up) beads	Beckman Coulter	A63881	Section 22
Q5 Hot Start DNA Polymerase	New England BioLabs	M0493	Section 23
5x Q5 Buffer (5x PCR Buffer)	New England BioLabs	B9027	Section 23
QIAquick Gel Extraction Kit	Qiagen	28704	Section 25
Qubit Fluorometer	Invitrogen	Q33216	Section 26
Optical Clear Qubit tubes	Invitrogen	Q32856	Section 26
Qubit dsDNA High Sensitivity Assay kit	Invitrogen	Q32851	Section 26