Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Summary

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Abstract

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.

ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.

Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo^{1, 2}. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Protocol

(The entire ChIP procedure takes approximately two days. Preparation of ChIP libraries for high-throughput sequencing takes another 2-3 days.)

1. Dissect and Prepare Tissue for ChIP Experiment (~1 million cells)

1. Dissect tissue of interest (e.g. 200 pairs of Drosophila testes) in cold PBS + 1x protease inhibitor (dissolve 1 pellet of protease inhibitor cocktail in 1.5 mL 1x PBS to obtain 7x stock solution) + PMSF (final concentration 100 μg/mL). Rinse tissue twice and resuspend tissue in 200 μL of the same PBS solution with inhibitors.
2. To fix the sample, add 5.5 μL 37% formaldehyde (warm formal....

Discussion

The versatility of ChIP analyses discussed in this protocol can be used on different tissues, which provides an opportunity to study the chromatin state in a biologically relevant system. ChIP experiments using cells from cultured systems are convenient to perform because large quantity of cells can be easily obtained. However, cultured cells do not necessarily reflect cells in a multi-cellular environment. By developing this technique using dissected tissue from live animals, we can address many questions that cultured .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Complete Mini protease inhibitor cocktail	Roche Group	11836153001
Formaldehyde (37%)	Supelco, Sigma-Aldrich	47083-U
PMSF	Sigma-Aldrich	78830
Kontes pellet pestle	Fisher Scientific	K749521-1590
PCR Purification Kit	Qiagen	28104
Linear polyacrylamide	Sigma-Aldrich	56575-1ML
Glycogen	Qiagen	158930
SYBR green/ROX qPCR Master Mix	Fermentas	K0223
Mini plate spinner	Labnet International	Z723533
Real time PCR system	Applied Biosystems	4351101
Small Volume Ultrasonic Processor	Misonix	HS-XL2000	Model discontinued
Dynabeads, Protein A	Invitrogen	100-01D
Dynamag magnet	Invitrogen	123-21D
Phenol:Chlorofrom:IAA	Invitrogen	15593-049
Epicentre DNA END-Repair Kit	Epicentre Biotechnologies	ER0720
MinElute Reaction Cleanup Kit	Qiagen	28204
Klenow Fragment (3’→5’ exo-)	New England Biolabs	M0212S
T4 DNA ligase	Promega Corp.	M1794
Adaptor oligonucleotides	Illumina	PE-400-1001
Paired-End Primer 1.0 and 2.0	Illumina	1001783 1001 784
E-Gel Electorphoresis system	Invitrogen	G6512ST
2X Phusion HF Mastermix	Finnzymes	F-531