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, 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.
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.
According to German animal care guidelines, no Institutional Animal Care and Use Committee (IACUC) approval was necessary to perform the chicken embryo experiments. According to the local guidelines, only experiments with chicken HH44 (18-day) embryos and older require IACUC approval. However, the embryos used in this study were all in earlier stages of embryonic development (i.e., HH19 (72 h)).
NOTE: The purpose of this protocol is to provide a detailed description of the ChIP assay so it can be effectively combined with qPCR or next-generation sequencing (i.e., ChIP-seq) to investigate histone modifications in low-abundance embryonic samples (ranging from 5 x 104 – 5 x 105 cells for each ChIP reaction) and different tissue types (Figure 1). The protocol should be applicable to investigating the binding profiles of transcription factors and co-activators (e.g., p300). However, due to the lower abundance of these regulatory proteins, larger cell numbers are likely to be required (5 x 105 – 5 x 106 cells for each ChIP reaction).
1. Preparation of Eggs and Microdissection of the Chicken SNT
NOTE: The procedure for microdissections technically differs from tissue to tissue and from animal model to animal model. This section describes in detail the dissection protocol used to obtain brachial SNT sections from stage-HH19 chicken embryos as an example.
2. Crosslinking Proteins to DNA: Day 1
NOTE: Perform all steps on ice unless stated otherwise.
3. Lysis and Sonication
4. Antibody Incubation
5. Preparation of Magnetic Beads: Day 2
6. Immunoprecipitation of the Chromatin
7. Wash, Elute, and Reverse the Crosslinks
8. Digest Cellular Protein and RNA: Day 3
9. ChIP-qPCR
NOTE: Perform DNA extraction and purification, as described in the Supplementary Materials.
NOTE: Verify sonication efficiency as described in the Supplementary Materials, to confirm that 200- to 500-bp DNA fragments are obtained (Figure 3).
NOTE: A critical step is to determine if the ChIP actually worked. If there are known genomic binding sites for the protein of interest, primers can be designed for quantitative PCR (qPCR) to determine if the known sites are specifically enriched in the ChIP DNA, in comparison with negative-control regions not expected to be bound by the candidate proteins. As an example, this work shows the ChIP-qPCR results obtained with ChIPs performed for H3K4me3 (active promoter histone mark) and H3K27me3 (inactive promoter histone mark) in SNT sections isolated from HH14 chick embryos ( Figure 4A).
10. ChIP-seq Library Preparation; End Repair: Day 4
11. ChIP-seq Library Preparation; Adenylate 3' Ends
12. ChIP-seq Library Preparation; Ligate Adapters
13. ChIP-seq Library Preparation; Amplification of DNA Fragments
14. ChIP-seq Library Preparation; Library Validation
NOTE: The validation of the library is performed using a DNA and RNA quality-control system. Preparation of the ladder: aliquot 1 µL of genomic DNA Ladder into the first tube/well and add 3 µL of sample buffer.
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 (Figure 3). Then, the ChIP and input DNAs were used for ChIP-qPCR analysis to measure the enrichments at loci expected to be either bound or unbound by the investigated histone modifications (Figure 4A). In the example shown in Figure 4A, H3K27me3 and H3K4me3 enrichment levels in HH19 SNT sections were evaluated by ChIP-qPCR around the promoter regions of SOX17 and SOX2, two genes that are inactive and active in the SNT, respectively. As expected, the SOX2 promoter was strongly marked by H3K4me3 but not by H3K27me3, while the SOX17 promoter showed the opposite pattern (Figure 4A). By evaluating a few loci, it is possible to estimate the quality of the ChIP without losing much DNA material for downstream ChIP-seq analysis. Once the quality of the ChIPs was confirmed, ChIP-seq libraries were prepared and sequenced. Representative loci are shown for each of the investigated chicken embryonic tissues: (i) profiles for H3K27me3 and H3K4me3 in the SNT of HH19 chick embryos around PAX7 (Figure 4B); (ii) profiles for H3K4me2 and H3K27ac in the maxillary prominences of HH22 chicken embryos around SNAI1 (Figure 4C); and (iii) profile for H3K27me3 in HH3-stage chicken embryos around the TBX3/TBX5 locus (Figure 4D). These ChIP-seq experiments clearly illustrate that our ChIP protocol can be used to generate epigenomic profiles from limited amounts of biological material in a broad range of embryonic tissues.
Figure 1: Schematic Overview of ChIP Protocol for Low-abundance Embryonic Samples Isolated from Chicken Embryos. Please click here to view a larger version of this figure.
Figure 2: Brief Overview of the SNT Microdissection Protocol. The transverse SNT segment is cut off at the brachial level of an HH19 chicken embryo. After trypsin treatment, the non-neural tissue is removed mechanically using forceps. Not, notochord; SNT, spinal neural tube; and SO, somite. Please click here to view a larger version of this figure.
Figure 3: Example of Optimal Sonication. A chicken SNT HH19 sample was sonicated for 11 cycles, with 30 s ON and 45 s OFF pulses at "high" amplitude using a sonicator. The crosslinks were reversed and the purified DNA was resolved on a 1.5% agarose gel. The optimal fragment size is observed after 11 cycles, ranging from 200 – 500 bp. Please click here to view a larger version of this figure.
Figure 4: Representative Examples of ChIP Analysis by ChIP-qPCR or ChIP-seq. (A) H3K27me3 (left) and H3K4me3 (right) levels at the indicated regions were measured by ChIP-qPCR analysis performed using SNT sections isolated from HH19 chick embryos. The SOX2 promoter region is active in the SNT and is thus marked by H3K4me3 but not by H3K27me3; the SOX17 promoter region is inactive in the SNT and is therefore enriched in H3K27me3 but not in H3K4me3; Chr6 Neg represents an intergenic region in chicken chromosome 6 and serves as a negative control. Error bars represent the standard deviation from three technical replicates. (B) ChIP-seq (H3K27me3 in magenta, H3K4me3 in blue, and input in red) profiles from the SNTs of HH14 chick embryos are shown around the PAX7 locus. (C) ChIP-seq (H3K4me2 in orange, H3K27ac in green, and input in red) profiles from the maxillary prominences of HH22 chick embryos are shown around the SNAI1 locus. (D) ChIP-seq (H3K27me3 in magenta and input in red) profiles from HH3 chick embryos are shown around the TBX3/TBX5 locus. Please click here to view a larger version of this figure.
Figure 5: Example of ChIP-seq Library Validation using the Automated RNA Quality-control System. The same ChIP-seq libraries were analyzed with the automated RNA quality-control system before (A) and after (B) primer dimer cleanup. Primer dimers can be seen as a weak ~135 bp band in (A). Please click here to view a larger version of this figure.
Steps | Problem | Possible reason | Solution |
1,2,3,4 5 and 11 | Low ChIP signal to noise ratios according to ChIP-qPCR and/or ChIP-seq | Tissue quality compromised. | Perform micro-dissections in cold conditions; otherwise chromatin integrity could be compromised. |
Sample loss after centrifugation of crosslinked samples. | Use DMEM medium with 10 – 20% serum or repeat centrifugation step and increase time to 10 min. | ||
Cross-linking duration. | Optimize the crosslinking time. Increased crosslinking times can facilitate the detection of certain proteins, such as co-activators and co-repressors that do not bind to DNA directly. | ||
Sample loss due to multiple washes in PBS. | Reduce the number of washes ; centrifugation steps can be repeated at 1,500 x g. | ||
Suboptimal sonication resulting in either too long or too short DNA sizes. | Perform a time course experiment to optimize sonication conditions. Optimal fragment sizes ranging from 200 to 500 bp | ||
Antibody not suitable for ChIP. | Use a different antibody against the endogenous protein or an antibody against a tag added to an exogenously expressed proteins (e.g. Flag, HA). | ||
12 | Low or no signals in qPCR, even for input DNA | Suboptimal primers | Check the primer efficiency and specificity; design new primers. |
Insufficient amount of DNA | Not enough starting material. Increase the amount of DNA used per qPCR reaction. |
Table 1: Troubleshooting Guide for Various Steps Involved in the Protocol.
Primers | Sequence |
SOX2-F | CCTTGCTGGGAGTACGACAT |
SOX2-R | GCCCTGCAGTACAACTCCAT |
SOX17-F | CCCTGAACTGTCATGTGTGG |
SOX17-R | CAAACAGTTGCCTTTGAGCA |
Chr6-neg-F | CCGTCAATCTCTGCTTGTGA |
Chr6-neg-R | TGGAATCTGCTTGTCACTGC |
Table 2: Primer Sequences for ChIP-qPCR.
Preincubation | ||
Temperature | Time | |
95 °C | 5 min | |
Amplification | ||
Temperature | Time | |
95 °C | 10 s | |
60 °C | 10 s | 45 |
72 °C | 10 s | Cycles |
Melting Curve | ||
Temperature | Time | |
95 °C | 5 s | |
65 °C | 1 min | |
Cooling | ||
40 °C | 30 s |
Table 3: PCR Conditions for ChIP-qPCR.
Initial denaturation | ||
Temperature | Time | |
95 °C | 3 min | |
Amplification | ||
Temperature | Time | |
98 °C | 20 s | |
60 °C | 15 s | 15 |
72 °C | 30 s | Cycles |
Final extension | ||
Temperature | Time | |
72 °C | 5 s | |
Cooling | ||
4 °C | hold |
Table 4: PCR Conditions for Amplification of DNA Fragments during ChIP-seq Library Preparation.
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 contexts (e.g., broad H3K4me3 promoter domains and super-enhancers)6,7,9,10,11,19. However, due to the inherent limitation of the ChIP assay, which typically requires millions of cells, most histone modification profiles have been generated in in vitro cell lines and cell types that can be sorted in vivo in large quantities4. Most recently, several ChIP-seq protocols have been described that are compatible with extremely low cell numbers, thus dramatically expanding the range of cell types and tissues in which epigenomic profiles can, in principle, be generated13,14,15,16. These new ChIP-seq protocols each rely on specific ad hoc library preparation methods that, at least in some cases, require non-standard equipment and/or reagents14,15,16.
Here, we describe a ChIP protocol that works with low to intermediate cell numbers (5 x 104 – 5 x 105 cells) obtained in vivo from different embryonic tissues10. To increase the sensitivity of our ChIP assays, we have eliminated several steps normally used during ChIP, such as sequential cell and nuclear lysis, which can result in a progressive loss of valuable material. Thus, after crosslinking, samples are treated with a single lysis buffer and are subsequently sonicated to minimize sample loss. Importantly, our ChIP protocol is compatible with qPCR analysis, thus enabling the epigenomic analysis of selected loci of interest. Furthermore, our ChIP protocol can be combined with standard ChIP-seq library preparation methods, thus being potentially useful to most laboratories with access to next-generation sequencing technology.
ChIP and ChIP-seq protocols can be used not only to investigate the binding profiles of histone modifications but also to uncover the binding sites of other important transcriptional regulators, such as transcription factors and co-activators (e.g., p300 and CBP)20. Typically, ChIPs (and ChIP-seq) for transcription factors and co-activators, which are not as abundant as histones, require considerably more starting material than ChIPs for histone marks. Thus, the described protocol might not necessarily work for most transcription factors or co-activators unless the number of starting cells is considerably increased (e.g., 5 x 105 – 5 x 106 cells) and/or highly specific and efficient antibodies are available. Nevertheless, with further optimization and the constantly improving library preparation methods, we anticipate that ChIP-seq from limited cellular material will become feasible for most regulatory proteins.
Although our ChIP and ChIP-seq protocols have been extensively validated for a variety of histone modifications and embryonic tissues, we would like to highlight some critical steps that we consider essential to generating high-quality epigenomic profiles. First, the embryonic samples need to be freshly isolated under conditions that do not compromise chromatin integrity. These include performing dissections under cold conditions or flash-freezing pooled samples until sufficient material is accumulated. Another critical step is sonication, which needs to be accurately optimized to perform high-quality ChIPs. Optimal sonication conditions frequently vary, depending on the tissue type, the amount of tissue, or the sonicator being used. Last but not least, a successful ChIP ultimately relies on the availability of specific and ChIP-compatible antibodies against the proteins of interest.
The authors have nothing to disclose.
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.
Reagent | |||
BSA powder | Carl Roth | 3737.3 | |
Phosphate Saline buffer (PBS) | Sigma Aldrich | D8537 | |
Tris-HCL pH8.0 | Sigma Aldrich | T1503 | |
NaCl | Carl Roth | 3957.2 | |
EDTA | Carl Roth | 8043.2 | |
EGTA | Carl Roth | 3054.2 | |
Na-Deoxycholate | Sigma Aldrich | D6750-24 | |
N-lauroylsarcosine | Sigma Aldrich | 61743-25G | |
Hepes | Applichem | A3724,0250 | |
LiCl | Carl Roth | 3739.2 | |
NP-40 | Sigma Aldrich | I3021-100ml | |
SDS | Carl Roth | 1833 | |
Protein G/magnetic beads | Invitrogen | 1004D | |
37% Formaldehyde | Sigma Aldrich | 252549-1L | |
Glycine | |||
RNase | Peqlab | 12-RA-03 | |
Proteinase K | Sigma Aldrich | 46.35 E | |
Na-butyrate | Sigma Aldrich | SLB2659V | |
Proteinase inhibitor | Roche | 5892791001 | |
SYBRgreen Mix | biozym | 617004 | |
dH2O | Sigma Aldrich | W4502 | |
1Kb ladder | Thermofisher | SM1333 | |
Orange G | Sigma Alrich | 03756-25 | |
Agarose | Invitrogen | 16500-500 | |
0.25% Trypsin-EDTA (1X) | Gibco | 25200-072 | |
Octylphenol Ethoxylate (Triton X 100) | Roth | 3051.4 | |
DMEM (Dulbecco´s Modified Eagle Medium) | Gibco | 31331-028 | |
Gel loading tips Multiflex | A.Hartenstein | GS21 | |
qPCR Plates | Sarstedt | 721,985,202 | |
384 well | Sarstedt | 721,985,202 | |
1.5 mL tubes | Sarstedt | 72,706 | |
100-1000µl Filter tips | Sarstedt | 70,762,211 | |
2-20µl Filter tips | Sarstedt | 70,760,213 | |
2-200µl Filter tips | Sarstedt | 70,760,211 | |
0.5-10µl Filter tips | Sarstedt | 701,116,210 | |
H3K27me3 antibody | Active Motif | 39155 | |
H3K27ac antibody | Active Motif | 39133 | |
H3K4me3 antibody | Active Motif | 39159 | |
H3K4me2 antibody | Active Motif | 39141 | |
End Repair Mix | Illumina | FC-121-4001 | |
Paramagnetic beads | Beckman Coulter | A63881 | |
Resuspension buffer | Illumina | FC-121-4001 | |
A-Tailing Mix | Illumina | FC-121-4001 | |
Ligation Mix | Illumina | FC-121-4001 | |
RNA Adapter Indexes | Illumina | RS-122-2101 | |
Stop Ligation Buffer | Illumina | FC-121-4001 | |
PCR Primer Cocktail | Illumina | FC-121-4001 | |
Enhanced PCR Mix | Illumina | FC-121-4001 | |
Genomic DNA ladder | Agilent | 5067-5582 | |
Elution Buffer | Agilent | 19086 | |
Sample Buffer | Agilent | 5067-5582 | |
Library Quantification Kit | Kapa Biosystems | KK4835 – 07960204001 | |
Fertile chicken white eggs | LSL Rhein Main | KN: 15968 | |
Needle (Neoject) | A.Hartenstein | 10001 | |
Syringe (Ecoject 10ml) | Dispomed witt oHG | 21010 | |
Name | Company | Catalog Number | コメント |
Equipment | |||
Centrifuge | Hermle | Z 216 MK | |
Thermoshaker | ITABIS | MKR13 | |
Sonicator | Active Motive | EpiShear probe sonicator | |
Sonicator | Diagenode | Bioruptor Plus | |
Rotator | Stuart | SB3 | |
Variable volume (5–1,000 μl) pipettes | Eppendorf | N/A | |
Timer | Sigma | N/A | |
Magnetic holder | Thermo Fisher | DynaMag-2 12321D | |
Table spinner | Heathrow Scientific | Sprout | |
Mixer | LMS | VTX 3000L | |
Real-Time PCR Cycler | Roche | Light Cycler; Serial Nr.5662 | |
PCR Cycler | Applied Biosystems | Gene Amp PCR System 9700 | |
DNA and RNA quality control system | Agilent | Agilent 4200 TapeStation System | |
Forceps | Dumont | 5-Inox-H | |
Perforated spoon | World precision instruments | 501997 |