An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

Summary

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

Abstract

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

Introduction

The majority of mammalian genomes (98 - 99%) are comprised of noncoding sequence, and these nocoding regions contain regulatory elements known to participate in controlling gene expression and chromatin organization^1,2. In a normal cell, the specific assembly of genomic DNA into compacted chromatin structure is critical for the spatial organization, regulation and precise timing of various DNA-associated processes^3,4,5. In a cancer cell however, chromatin modifications by aberrant epigenetic mechanisms can lead to imp....

Protocol

All clinical specimens were obtained following the guidelines of Institutional Review Board.

1. Buffer Preparation

1. Make 200 mL of TE buffer (10 mM Tris-HCl, 10 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0).
2. Make 200 mL of STE buffer (10 mM Tris-HCl, 10 mM EDTA pH 8.0, 140 mM NaCl).
3. Make 200 mL of 2.0 M glycine (37.52 g of glycine in 200 mL of water) and heat it to 65 °C.
4. Make 200 mL of 5% sodium deoxycholate (DOC) solution (10 g of DOC in 200 mL of water).
5. Make 500 mL of ChIP harvest buffer (12 mM Tris-Cl, 0.1x phosphate buffered saline (PBS), 6 mM EDTA, 0.5% sodium dodecyl sulfat....

Results

This protocol allows the immunoprecipitation from frozen tumor tissues and cell lines that can be performed on dozens of samples in parallel using a high-throughput method (Figure 1A). Chromatin fragments should range between ~200 - 1000 bps for optimal immunoprecipitation. We have noted that the time needed to achieve same shearing length differs for different tissue and cell types. The success of ChIP from small amounts of tissue depends on.......

Discussion

This protocol describes a complete and comprehensive high-throughput ChIP-seq module for genome-wide mapping of chromatin states in human tumor tissues and cell lines. In any ChIP-seq protocol, one of the most important steps is antibody specificity. Here, this method illustrates immunoprecipitation conditions for the described six histone modifications, all of which are ChIP-grade and have been previously validated in our and other laboratories^42,44,<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ChIP-grade H3K4me1 antibody	Abcam	ab8895
ChIP-grade H3K27ac antibody	Abcam	ab4729
ChIP-grade H3K4me3 antibody	Abcam	ab8580
ChIP-grade H3K79me2 antibody	Abcam	ab3594
ChIP-grade H3K27me3 antibody	Abcam	ab6002
ChIP-grade H3K9me3 antibody	Abcam	ab8898
1M Tris HCl, pH 8.0	Teknova	T1080
EDTA	Sigma-Aldrich	E9884
NaCl	Sigma-Aldrich	S7653
Glycine	Sigma-Aldrich	G8898
Sodium deoxycholate	Sigma-Aldrich	30970
DPBS	Sigma - Life Sciences	D8537-500ML
SDS	Sigma-Aldrich	74255
Triton-X	Sigma-Aldrich	X100-100ML
LiCl	Sigma-Aldrich	746460
NP-40	Calbiochem	492016-100ML
1% TWEEN-20	Fisher Bioreagents	BP337-500
BSA - IgG-free	Sigma - Life Sciences	A2058-5G
HBSS	Gibo	14025092
GentleMACS C tube	GentleMACS	120-008-466	disassociation tube
16% Formaldehyde	Peierce	28906
miniProtease inhibitor	Roche Diagnostics	11836153001	protease inhibitor tablets
Dynabeads Protein G	Invitrogen	10009D
Bioruptor NGS tubes 0.65 mL	Diagenode	C30010011	sonication tubes
DynaMag - 96 Side Skirted	Invitrogen	120.27	96-well magnetic stand
TE buffer	Promega	V6231
RNase A	Invitrogen	12091021
Proteinase K	Invitrogen	100005393
AMPure XP beads	Beckman Coulter	A63882	paramagnetic beads
Ethanol	Sigma-Aldrich	E7023
Qubit ds DNA High Sensitivity Assay Kit	Invitrogen	Q32854	high sensitivity DNA reagents
NEBNext Ultra II DNA Library Prep Kit	New England BioLabs	E7645L	DNA Library Prep Kit
Nuclease-free water	Ambion	AM9932
High sensitivity D1000 ScreenTape	Agilent Technologies	5067-5584	high sensitivity DNA reagents
High sensitivity D1000 reagents	Agilent Technologies	5067-5585	high sensitivity DNA reagents
Multiplex Oligos (Index primers- Set 1)	New England BioLabs	E7335L	Multiplex Oligos
Multiplex Oligos (Index primers- Set 2)	New England BioLabs	E7500L	Multiplex Oligos
TapeStation 4200	Agilent Technologies	G2991AA	high sensitivity DNA electropherogram instrument
Bioruptor Pico sonication device	Diagenode	B01060001	water bath disruputor
Mixer	Nutator	421105
Bio-Rad C1000 Touch Thermal Cycler	Bio-Rad	1851196	PCR Thermal cycler
Water Bath	Fisher Scientific	2322
Multichannel Pipet	Denville	1003123
Tube Revolver	Thermo-Scientific	88881001
96-Well Skirted Plate	Eppendorf	47744-110
Allegra X-12R Centrifuge	Beckman Coulter	A99464	benchtop centrifuge
Centrifuge 5424	Eppendorf	22620461	tabletop centrifuge
Optical tube strips (8x Strip)	Agilent Technologies	401428
Optical tube strip caps (8x strip)	Agilent Technologies	401425
Loading Tips, 10 Pk	Agilent Technologies	5067-5599
IKA MS3 vortex	IKA	3617000	vortex