Name: Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis (Video) | JoVE Journal
Uploaded: 2017-12-12T12:30:00.000+00:00
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Description: Learn native ChIP-seq for nucleosome density analysis using MNase accessibility and histone modification measurements. This method reveals epigenomic features at single nucleosome resolution in diverse cell types.

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A.L. was supported by a Canadian Graduate Scholarship from the Canadian Institutes of Health Research. This work was supported by grants from Genome British Columbia and the Canadian Institutes of Health Research (CIHR-120589) &#160;as part of the Canadian Epigenetics, Environment and Health Research Consortium Initiative and by the Terry Fox Research Institute Program Project Grant #TFF-122869 to M.H. and a Terry Fox Research Institute New Investigator Award (Grant # 1039) to M.H.

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The authors declare that they have no competing financial interests.

Given the combinatorial nature of chromatin modification and nucleosome positioning in transcriptional regulation, a method that enables simultaneous measurements of these features is likely to provide new insights into epigenetic regulation. The ndChIP-seq protocol presented here is a native ChIP-seq protocol optimized to enable simultaneous interrogation of histone modification and nucleosome density in rare primary cells9,10. ndChIP-seq utilizes enzymatic digestion of chromatin that, when coupled to paired-end massively parallel sequencing and a Gaussian mixture distribution model, allows for the investigation histone modifications at single nucleosome level and the deconvolution of epigenomic profiles driven by heterogeneity within a population. Using this protocol, we have previously reported a unique distribution of immuno-precipitated fragment sizes, determined by paired-end read boundaries, associated with specific chromatin states defined by ChromHMM10,24.
The quality of a ndChIP-seq library depends on multiple factors, such as antibody specificity and sensitivity, optimal MNase digestion conditions, and quality of the chromatin. The specificity of the antibodies used is crucial in producing successful ndChIP-seq library. An ideal antibody shows high affinity against the epitope of interest with little cross reactivity with other epitopes. It is equally important to choose magnetic beads with the highest affinity for the antibody of choice.
MNase digestion is a critical and time- and concentration-sensitive reaction in this protocol. Therefore, when processing multiple samples, it is important that each reaction is incubated for an equivalent amount of time (see step 2.2). The quality of the chromatin is another factor that significantly effects the outcome of ndChIP-seq. Fragmented chromatin leads to a sub-optimal MNase digestion profile and results in libraries with a low signal to noise ratio. Primary samples with low cell viability or degraded chromatin, such as formalin-fixed paraffin-embedded (FFPE) tissue are not recommended for this protocol.
The addition of a PIC during chromatin extraction reduces undesired (i.e., random) chromatin fragmentation and preserves integrity of histone tails. As such, PIC needs to be added to lysis buffer and immunoprecipitation buffer just prior to use. While selecting cells via flow cytometry, select for viable cells and ensure cells are sorted at a low flow rate to increase the accuracy of the cell number estimate and viability of cells. Avoid sorting cells directly into lysis buffer. The sheath buffer will dilute the lysis buffer and prevents effective permeabilization of the cell membrane to MNase. Depending on a type of cells or organism, titration of MNase may be required to obtain optimal digestion.
ndChIP-seq on mammalian cells requires a minimum sequencing depth of 50 million paired-reads (25 million fragments) for narrow marks and 100 million paired-reads (50 million fragments) for broad marks and input. The Gaussian mixture distribution algorithm will not perform optimally on libraries that have been sequenced to a depth significantly below this recommendation. ndChIP-seq will not classify promoters with little separation between the weighted distribution value for mono- and di-nucleosome fragment lengths into mono- or di-nucleosome dominated promoters. Therefore, these promoters must be removed in the subsequent analysis. Biological replicates can be generated to increase confidence in predicted distributions and identify technical variability in the MNase digestion and library construction.
Unlike previous iterations of native ChIP-seq protocols, ndChIP-seq provides the means to investigate combinatorial effect of chromatin structure and histone modification by utilizing fragment size post immunoprecipitation to integrate nucleosome density, determined by MNase accessibility, with histone modification measurements. Application of ndChIP-seq to primary cells and tissues will provide novel insights into the integrative nature of epigenetic regulation and permit identification of epigenetic signatures due to heterogeneity within the population.

The eukaryotic genome is packaged into chromatin via repeating nucleosome structures that consist of two copies of four histone proteins (e.g., H2A, H2B, H3, and H4) circumscribed by 146 base pairs of DNA1,2. Chromatin remodeling complexes control nucleosome position within gene promoter boundaries and participate in the regulation of gene expression by altering accessibility of the DNA to transcription factors and to the RNA polymerase machinery3,4.
Amino terminal tails of histones within the nucleosome are subjected to various covalent modifications, including acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, formylation, and hydroxylation of specific amino acids5,6,7,8. Positions and degrees of these modifications dictate a chromatin state that influence chromatin structure and control access of the molecular complexes that allow activation of transcription7. Given that both nucleosome density and histone modification play a role in the local control of gene transcription, we developed a native ChIP approach that enables the simultaneous measurement of nucleosome density and histone modification9,10.
Native ChIP-seq takes advantage of the endonuclease micrococci nuclease (MNase) to digest intact chromatin in its native state within the nucleus11,12, a property that has been leveraged to map nucleosome positioning13,14,15. Nucleosome density ChIP-seq (ndChIP-seq) takes advantage of the property of preferential access of MNase to open regions of chromatin to generate measurements that combine MNase accessibility with histone modification10. ndChIP-seq is suitable for the profiling of histone modifications in rare primary cells, tissues, and cultured cells. Here, we present a detailed protocol that enables the generation of sequence datasets suitable for a previously described analytical frame work10 that integrates fragment size post immunoprecipitation, determined by paired-end read boundaries, to simultaneously investigate MNase accessibility with histone modification measurements. Previously, application of this protocol to 10,000 primary human cord blood derived CD34+ cells and human embryonic stem cells revealed unique relationships between chromatin structure and histone modifications within these cell types10. Given its ability to simultaneously measure nucleosome accessibility and histone modification, ndChIP-seq is capable of revealing epigenomic features in a cell population at a single nucleosome level, and resolving heterogeneous signatures into their constituent elements. An example of the exploration of heterogeneous cellular populations by ndChIP-seq is investigation of bivalent promoters, where both H3K4me3, an active mark, and H3K27me3, a repressive mark, are present10.

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generation of native chromatin immunoprecipitation sequencing libraries for nucleosome density analysis

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

The overall goal of this procedure is to generate native chromatin immunoprecipitation sequencing libraries for nucleosome density analysis. This method can answer questions in epigenetics such as revealing epigenomic features in a cell population at a single nucleosome level and resolving heterogeneous chromatin signatures into their continuous elements. The main advantage of this technique is to utilize MNase property of preferential access to open chromatin region to generate a measurement that combines MNase accessibility with histone modification.Generally, individuals new to this method will struggle because they are not able to obtain optimal MNase digestion. Begin this protocol with cell preparation as described in the text protocol. On day one, thaw each cell pellet in a 37-degrees-Celsius water bath for 10 seconds.Then, transfer the cells to ice. To each cell pellet, immediately add an ice-cold one-X lysis buffer plus one-X PIC to a final concentration of 10, 000 cells per 20 microliters. Mix 10 times by pipetting up and down without creating bubbles.Working on ice, aliquot 20 microliters per well of the resulting lysates into a 96-well plate. Cover the plate with the plastic seal before incubating on ice for 20 minutes. Label the plate as MNase and record the wells to a template key.Just before 20-minute lysis is complete, dilute the MNase one enzyme with MNase one dilution buffer to a final concentration of 20 units per microliter and keep it on ice. Working on ice, prepare the MNase one digestion master mix as described in the text protocol. Then, aliquot 20 microliters of the master mix per each row of samples, plus five microliters of extra volume into a 96-well reservoir plate.After the lysates finish incubating, remove the MNase digestion plate from the ice. Using a multi-channel pipette, add 20 microliters of MNnase one digestion master mix into each row of samples and mix by pipetting up and down 10 times. Change the tips between rows.Then, incubate the plate at room temperature for exactly five minutes. To stop the reaction after five minutes, use a multi-channel pipette to add six microliters of 250-micromolar EDTA into each row of samples and mix up and down a few times. Make sure to change tips between rows.After EDTA addition, switch the setting of the pipette to 20 microliters, and mix 10 times as before to assure complete stop of the digestion reaction. Finally, add six microliters of 10X lysis buffer to each row of the MNase digested samples and mix well by pipetting up and down 10 times. Cover the plate with a plastic seal and incubate on ice for 15 minutes.Prepare the antibody bead complex plate and the pre-clearing plate as described in the text protocol. Quick-spin both plates by centrifuging for 10 seconds at 200 times G.Then, place the antibody bead complex plate on a plate magnet and wait 15 seconds for the solution to become clear. Carefully remove and discard the supernatant using a pipette without disturbing the beads.Then, remove the plate from the plate magnet and keep it on ice. Place the pre-clearing reaction plate on a plate magnet and wait 15 seconds for the beads to separate and for the solution to become clear. Without disturbing the beads, use a pipette to carefully transfer the supernatant to the corresponding wells of the antibody bead complex plate kept on ice.Mix each well gently by pipetting up and down 15 times. Seal the plate well with an aluminum plate cover and incubate overnight at four degrees Celsius on a rotating platform. Following incubation, relabel the plate IP Reaction.On day two, set the heating mixer to 65 degrees Celsius, then prepare a low-salt wash buffer and high-salt wash buffer on ice. Quick-spin the IP reaction plate for 10 seconds at 200 times G.Place the IP reaction plate on a plate magnet and wait 15 seconds for the solution to become clear. Using a multi-channel pipette, without disturbing the beads, carefully remove and discard the supernatant.Take the plate off the plate magnet and place it on ice. To each row of samples in the IP reaction plate, add 150 microliters of ice-cold low-salt wash buffer and mix slowly up and down for 10 times to fully resuspend the beads. Place the IP reaction plate back onto the plate magnet and remove the supernatant as before.Then, place the plate back on ice and repeat the wash steps for a total of two washes. Working on ice, add 150 microliters of the high-salt wash buffer to each row of samples in the IP reaction plate. Mix the samples slowly by pipetting up and down 10 times to fully resuspend the beads.After removing the supernatant as before, place the IP reaction plate on ice and prechill a new 96-well plate beside it. To each row of samples in the IP reaction plate, add 150 microliters of the high-salt wash buffer and mix slowly 10 times by pipetting up and down to fully resuspend the beads. After resuspension, transfer each row of samples to the corresponding row of the new, pre-chilled 96-well plate.Discard the old plate. Place the new IP reaction plate on the plate magnet and discard the supernatant as before. Keep the plate at room temperature.To each row of samples in the IP reaction plate, add 30 microliters of the ChIP elution buffer and mix slowly by pipetting up and down 10 times. Take care to prevent bubble formation. Seal the plate with a PCR cover and incubate in a heating mixer at 65 degrees Celsius for 1 1/2 hours with a mixing speed of 1, 350 RPM.After a 1 1/2-hour incubation, spin down the IP reaction plate at 200 times G for one minute at four degrees Celsius. Then, place the IP reaction plate on a plate magnet and wait for the solution to clear. Using a multi-channel pipette, without disturbing the beads, carefully transfer 20 microliters of the supernatant into a new 96-well plate using fresh tips for each row.Label the plate IP Reaction and keep it at room temperature. Proceed to protein digestion followed by library construction as described in the text protocol. Shown here are representative results of optimal, under-digested and over-digested MNase digested chromatin before library generation.The optimally-digested profile is dominated by single nucleosome fragment sizes while not over-digested to allow for recovery of higher order nucleosome fragments. Suboptimal digestion of chromatin will also be apparent in the profile of the sequencing library generated from the IP material. To validate the libraries by qPCR, fold enrichment of IP libraries with respect to input library was assessed for positive and negative targets.In addition, inspection of the aligned reads of libraries with high qPCR assessed fold enrichment on a genome browser should reveal visually detectable enrichments as compared to the input library. Successful nucleosome density ChIP-sequencing libraries will contain highly-correlated replicates with a significant portion of aligned reads within MACS2-identified enriched peaks. Once mastered, this technique can be done in two days if it is performed properly.After watching this video, you should have a good understanding of how to generate native chromatin immunoprecipitation sequencing libraries for nucleosome density analysis. While attempting this procedure, it is important to remember to make sure that the chromatin is optimally digested and only use antibody that show a high affinity with the epitope of interest and show little or no cross-reactivity with other epitopes. Following this procedure, computational modeling of MNase accessibility can be performed to answer questions such as epigenetic signatures due to heterogeneity within a cell population.This method provides insight into combinatorial effect of nucleosome position and local density as well as post-translational modification of their histone subunits and it can be applied to any systems such as human or mouse, primary cells and frozen tissues.

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Research

JoVE Journal

Genetics

In this video, we demonstrate the chromatin immunoprecipitation technique to identify protein binding sites on specific regions of the genomic DNA of oligodendrocyte precursor cells via the selective immunoprecipitation of protein-bound chromatin fragments. This method helps to study the interaction of several regulatory proteins, including transcription factors involved in gene regulation.

Chromatin Immunoprecipitation to Identify Target Protein Binding Sites on Genomic DNA

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)

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research but they are laborious, costly, and time consuming. Here a commercially available robotic liquid handling system that automates chromatin immunoprecipitation for mapping in vivo protein-DNA interactions with limited amounts of cells is presented.

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

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) Protocol for Low-abundance Embryonic Samples

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

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.

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

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

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

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

We present a three-color chromatin single molecule localization microscopy (SMLM) staining and analysis protocol that enables reproducible mapping of euchromatin, heterochromatin, and RNAP II for spatial analysis. This protocol enables efficient multicolor labeling in dense nuclear environments, including chromatin-associated targets, allowing reliable simultaneous detection.

A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

Learn native ChIP-seq for nucleosome density analysis using MNase accessibility and histone modification measurements. This method reveals epigenomic features at single nucleosome resolution in diverse cell types.

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

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