Name: Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples (Video) | JoVE Journal
Uploaded: 2017-08-29T14:00:00.000+00:00
Duration: 12 min 47 s
Description: Learn chromatin immunoprecipitation for epigenomic profiling using low-abundance chicken embryonic samples. This method enables researchers to study histone modifications and transcription regulation in developmental biology.

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5 minutes

The authors thank Jan Appel for his excellent technical assistance during the establishment of this protocol. Work in the Rada-Iglesias laboratory is supported by CMMC intramural funding, DFG Research Grants (RA 2547/1-1, RA 2547/2-1, and TE 1007/3-1), the UoC Advanced Researcher Group Grant, and the CECAD Grant.

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<li>Creyghton, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+H3K27ac+separates+active+from+poised+enhancers+and+predicts+developmental+state%5BTitle%5D)+AND+%22Proc+Nat+Acad+Sci+USA%22%5BJournal%5D)">Histone H3K27ac separates active from poised enhancers and predicts developmental state.</a> Proc Nat Acad Sci USA. 107 (50), 21931-21936 (2010).</li>
<li>Benayoun, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((H3K4me3+breadth+is+linked+to+cell+identity+and+transcriptional+consistency%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">H3K4me3 breadth is linked to cell identity and transcriptional consistency.</a> Cell. 158 (3), 673-688 (2014).</li>
<li>Rehimi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenomics-Based+Identification+of+Major+Cell+Identity+Regulators+within+Heterogeneous+Cell+Populations%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Epigenomics-Based Identification of Major Cell Identity Regulators within Heterogeneous Cell Populations.</a> Cell Rep. 17 (11), 3062-3076 (2016).</li>
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<li>Schmidl, C., Rendeiro, A. F., Sheffield, N. C., Bock, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ChIPmentation%3A+fast%2C+robust%2C+low-input+ChIP-seq+for+histones+and+transcription+factors%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors.</a> Nat Methods. 12 (10), 963-965 (2015).</li>
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The authors do not have any competing financial interests to be disclosed.

Epigenomic profiling of histone modification using ChIP-seq can be used to improve the functional annotation of vertebrate genomes in different cellular contexts4,5,18. These epigenomic profiles can be used, among other things, to identify enhancer elements, to define the regulatory state of enhancers (i.e., active, primed, or poised), and to define major cell identity regulators in different biological or pathological ...

Histone post-translational modifications are directly involved in various chromatin-dependent processes, including transcription, replication and DNA repair1,2,3. Moreover, different histone modifications show positive (e.g., H3K4me3 and H3K27ac) or negative (e.g., H3K9me3 and H3K27me3) correlations with gene expression and can be broadly defined as activating or repressive histone marks, respectively2,3. Consequently, global histone modification maps, also referred to as epigenomic maps, have emerged as powerful and universal tools to functionally annotate vertebrate genomes4,5. For example, distal regulatory sequences such as enhancers can be identified based on the presence of specific chromatin signatures (e.g., active enhancers: H3K4me1 and H3K27ac), which distinguish them from proximal promoter regions (e.g., active promoters: H3K4me3)6,7,8. On the other hand, genes with major cell identity regulatory functions are typically found with broad chromatin domains marked with H3K4me3 or H3K27me3, depending on the transcriptionally active or inactive status of the underlying genes, respectively9,10. Similarly, the expression of major cell identity genes seems to be frequently controlled by multiple and spatially clustered enhancers (i.e., super-enhancers), which can be identified as broad H3K27ac-marked domains11.
Currently, histone modification maps are generated using ChIP-seq technology, which in comparison to previous approaches such as ChIP-chip (ChIP coupled to microarrays) provides higher resolution, fewer artifacts, less noise, greater coverage, and lower costs12. Nevertheless, the generation of epigenomic maps using ChIP-seq technology has its inherent limitations, mostly associated with the capacity to successfully perform ChIP in the samples of interest. Traditional ChIP protocols typically required millions of cells, which limit the applicability of this method to in vitro cell lines or cells that can easily be isolated in vivo (e.g., blood cells). In the last few years, a number of modified ChIP protocols compatible with low cell numbers have been described13,14,15,16. However, these protocols are specifically designed to be coupled with next-generation sequencing (i.e., ChIP-seq), and they typically use ad hoc library preparation methods13,14,15,16.
Here, we described a ChIP protocol that can be used to investigate histone modification profiles using low to intermediate cell numbers (5 x 104 - 5 x 105 cells) at either selected loci (i.e., ChIP-qPCR) or globally (i.e., ChIP-seq) (Figure 1). When coupled to ChIP-seq technology, our ChIP protocol can be used together with standard library preparation methods, thus making it broadly accessible to many laboratories10. This protocol has been used to investigate several histone marks (e.g., H3K4me3, H3K27me3, and H3K27ac) in different chicken embryonic tissues (e.g., spinal neural tube (SNT), frontonasal prominences, and epiblast). However, we anticipate that it should be broadly applicable to other organisms in which biologically and/or clinically relevant samples can be only obtained in low amounts.

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chromatin immunoprecipitation (chip) protocol for low-abundance embryonic samples

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

To illustrate the performance of our ChIP protocol, we performed ChIP-seq experiments using pooled SNT sections from HH19 chicken embryos, maxillary prominences of HH22 chicken embryos, and stage-HH3 chicken embryos to investigate the binding profiles of various histone modifications (i.e., H3K4me2, H3K27ac, H3K4me3, and H3K27me3). Once ChIP DNAs were obtained, the sonication efficiency was evaluated by agarose gel electrophoresis of the corresponding input DNAs (<strong class="x...

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

The overall goal of this procedure is to investigate the binding profiles of different histone modifications, using low-abundance embryonic samples in order to improve the functional annotation of vertebrate genomes. This method can help answer key questions in the developmental biology field such as, which are the key enhancers and genes defining the identity of a particular embryonic tissue. The main advantages of this techniques are that it requires a limited amount of particular material and it can also be used for both local specific and genome wide analysis.Though this method can provide insights into transcription regulation or cellular heterogeneity with the embryonic tissues, it can also be applied to other systems such as, patent samples. To obtain stage HH19, chicken embryos for this experiment, incubate fertile white Leghorn hen eggs in horizontal orientation at 37 degrees celsius and 80%humidity for three days. To start isolating the HH19 embryos, make them accessible by extracting three milliliters of albumin with a 10 millimeter syringe to lower the blastoderm.In order to remove extra embryonic membranes, use fine scissors to make a small opening in the eye. Add one milliliter of lock solution. With the help of a bended needle, add 0.5 milliliters of Indian black ink.Use fine surgical scissors and forceps to cut off the extra embryonic membrane that surrounds the embryo. Then, transfer the embryo with a perforated spoon to a 4.5 centimeter petri dish containing 20 milliliters of PBS solution. Working on ice under a stereo microscope, use fine scissors and forceps to dissect and discard the amnion and the remaining parts of extra embryonic membrane.Then, cut the transverse spinal neuro tube or SNT segment at the brachial level of the embryo. Extend the dissection caudally into the thoracic region to isolate the SNT. To finish, use forceps to remove the tissues that laterally and ventrally surround the SNT.After isolating SNTs, immerse each segment in a 3.5 centimeter petri dish containing 37 degree celsius warm trypsin. When loosening of the non-neuro tissues around the SNT is visible under a stereo microscope, transfer the samples into cold 1x PBS. To stop the trypsinization, transfer the samples into a new dish with cold DMEM containing 10%FBS.Use forceps to manually remove the remaining mesenchymal and ectodermal tissues from the SNT. Transfer this clean SNT section to a 1.5 milliliter tube and flash freeze it in liquid nitrogen. Next, homogenize the tissue according to the text protocol.Add 13.5 microliters of 37%formaldehyde and incubate at room temperature on a rotator for 15 minutes. To quench the formaldehyde, add 25 microliters of 2.5 molar glycine to the tube and incubate at room temperature on a rotator for 10 minutes. Centrifuge the tube and then, wash the pellet according to the text protocol.After crosslinking, re-suspend the pellet in 300 microliters of ice cold, complete lysis buffer by pipetting. Incubate the samples at four degrees celsius on a rocking platform for 10 minutes. Next, sonicate the samples at four degrees celsius.After sonication, centrifuge the samples at 16, 000 x G at four degrees celsius for 10 minutes. Transfer the supernatant or lysate to a fresh 1.5 milliliter tube and discard the cellular debris pellet. Dilute the lysate according to the text protocol.Then, keep a small aliquot of each sample as input control and freeze at 20 degrees celsius. Incubate the remaining samples that will be subjected to chromatin precipitation or chip with appropriate antibodies according to the text protocol. Then, add 300 microliters of antibody bound chromatin to previously washed magnetic beads.Invert the tubes to mix the samples. To bind the antibodies to the beads, place the tubes on a tube rotator and rotate vertically at four degree celsius for a minimum of four hours. To wash the chromatin bound beads, add one milliliter of ice cold RIPA wash buffer to the tube kept on ice and mix by inverting or flicking the tube.After placing the tube into the magnetic holder at room temperature, wait for the beads to settle on the side. Pour off the clear liquid and repeat this wash three more times. After adding one milliliter of TE 50 millimolar sodium chloride to the tube, transfer the chromatin bound beads in this solution to a fresh 1.5 millimeter microcentrifuge tube.Place the tube into the magnetic holder and when the beads have settled, pour off the liquid. Next, centrifuge the beads at 900 x G at four degrees celsius for three minutes in a pre-cooled centrifuge. After the beads have been settled in the magnetic holder, remove all remaining TE using a gel loading tip.Working at room temperature, add 210 microliters of elution buffer to the beads and re-suspend by flicking. Incubate the tube in a pre-warmed heat block at 65 degree celsius for 15 minutes, while shaking. After incubation, centrifuge the tubes with the beads at 16, 000 x G at room temperature, for one minute.Once the beads have settled in the magnetic holder, transfer the supernatant to a fresh microcentrifuge tube. Thaw the previously frozen input control sample from 20 degrees celsius to room temperature. Add 90 microliters of elution buffer to the sample and mix by briefly vortexing.To reverse the cross-links, incubate the chip and control samples at 65 degrees celsius overnight. Working at room temperature, add one volume of TE buffer per tube and invert to mix. To digest the RNA, add RNAse A to the control and chip samples to a final concentration of 0.2 milligrams per milliliter and invert the tubes to mix.Incubate the tubes in a pre-warmed heat block at 37 degrees celsius for two hours. To digest the protein for the subsequent DNA purification, add protease K to a final concentration of 0.2 milligrams per milliliters to the tubes and invert the tubes to mix. Incubate the tubes in a pre-warmed heat block at 55 degrees celsius for two hours.Extract and purify the DNA according to the supplementary materials. This DNA can now be used for quantitative PCR and chip seek library preparation. Sonication efficiency essential for a successful chip is confirmed by agarose gel electrophoresis of the input control samples.In this example, using chicken spinal neural tube or SNT sections, 11 sonication cycles were used to obtain an optimal fragment size ranging from 200 to 500 base pairs. Chipped DNA was analyzed by quantitative PCR to measure the enrichment levels of activating and repressive histone marks around the promoter regions of genes that are active and inactive in the chicken SNT. This shows that it is possible to evaluate the chipped quality by quantifying only a few low side without losing much DNA for further analysis.After chip seek libraries were prepared in sequence, a representative locust is shown for H3K27 tri methylation and H3K4 tri methylation chip seek data generated in chicken SNT. Similar chip seek profiles were also successfully generated in different chicken embryonic tissues, such as maxillary prominences and stage HH3 embryos. This demonstrates that here presented, chip protocol can be used to generate epigenomic profiles from limited amounts of various embryonic tissues.By using this protocol to generate the histone modification profiles in an embryonic tissue, it is important to remember that signals will come from all different cell types present in such tissue. Following this protocol and using more starting biological material, it should be possible to investigate the binding profiles of other proteins involved in transcriptional regulation, such as, transcription factors, co-activators or RNA polymerase 2 sub units. Once established, these protocols will enable researchers in the field of developmental biology or medical genetics to improve the functional alteration of vertebrate genomes and to gain important insights into the molecular basis of myogenesis, evolution and human disease.

chromatin-immunoprecipitation-chip-protocol-for-low-abundance

Research

JoVE Journal

Genetics

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation. <br...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq—the most popular approach to investigate chromatin features at a global level—is outlined here for Xenopus embryos.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

This protocol focuses on chromatin preparation from snap frozen tissues and it is suitable for Crosslinking Chromatin Immunoprecipitation (X-ChIP) followed by either quantitative PCR analysis (X-ChIP-qPCR) or next generation sequencing approaches (X-ChIP-seq).

Chromatin Extraction from Frozen Chimeric Liver Tissue for Chromatin Immunoprecipitation Analysis

Learn chromatin immunoprecipitation for epigenomic profiling using low-abundance chicken embryonic samples. This method enables researchers to study histone modifications and transcription regulation in developmental biology.

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

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