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1. Native chromatin immunoprecipitation (ChIP)<ol><li>Chromatin preparation<ol style='list-style-type:decimal;'><li>After making stock of derived neurosphere cells (NS), culture NS in a T-75 flask in 15 mL of NSC medium at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2&#160;until NS become confluent. One confluent T-75 plate will yield 3-5 X 106&#160;cells.</li><li>When the cells become confluent, centrifuge NS at 300 x g for 5 min, decant the supernatant, and add 1 mL of cell dissociation medium. Incubate the cells at 37 &#176;C for 5 min. Add 5 mL of medium and count the cells using a hemocytometer. NOTE: 1 x 106&#160;cells are needed per immunoprecipitation (IP) reaction. Note that a pre-immune serum or IgG control must also be included.</li><li>Centrifuge the NS in one 1.5 mL microcentrifuge tube at 300 x g for 5 min, decant the supernatant and resuspend NS pellet with 1 mL of balanced salt solution and transfer the suspension to low protein binding 1.5 mL microcentrifuge tubes.</li><li>Centrifuge NS at 300 x g for 5 min, decant the supernatant and re-suspend the cell pellet in 95 &#181;L of digestion buffer (50 mM Tris-HCl, pH 8.0, 1 mM CaCl2, 0.2% polyethylene glycol octylphenyl ether) per 1 x 106&#160;cells supplemented with protease inhibitor cocktail at a high concentration (1:100). Immediately pipet the cells up and down to prevent clumping and avoid creating any bubbles.</li><li>Resuspend micrococcal nuclease (MNase) in 50% glycerol to make 1 mg/mL solution (1.15 units/&#181;L, stored at -20 &#176;C) that will be referred to as "1x" stock. Make a "0.1x" stock by doing a second 1:9 dilution with 50% glycerol (e.g., 900 &#181;L of "1x" MNase and 100 &#181;L of 50% glycerol) and store it at -20 &#176;C.</li><li>Mix 5 &#181;L of "0.1x" MNase and 145 &#181;L of digestion buffer. Keep the enzyme on ice all the time. Add 5 &#181;L of diluted MNase in digestion buffer per 1 x 106&#160;cells. Flick the tube to mix and place the tube in 37 &#176;C block for exactly 12 min. Process the samples one at a time to ensure an accurate incubation time of 12 min.</li><li>Add 10 &#181;L of 10x MNase Stop Buffer (110 mM Tris-HCl, pH 8.0, 55 mM Ethylenediaminetetraacetic acid (EDTA)) per 1 x 106&#160;cells. Samples must be kept on ice from this point onwards.</li><li>Add 110 &#181;L "2x Radioimmunoprecipitation Assay buffer (RIPA) buffer" (280 mM NaCl, 1.8% polyethylene glycol octylphenyl ether, 0.2% sodium dodecyl sulfate (SDS), 0.2% Na-deoxycholate, 5 mM ethylene glycol-bis (&#946;-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); stored at 4 &#176;C) supplemented with protease inhibitor cocktail at high concentration (1:100) per 1 x 106&#160;cells. Flick the tube to mix.</li><li>Centrifuge the tubes for 15 min in a microfuge at 4 &#176;C and 1700 x g. Transfer the supernatant to a new regular 1.5 mL microcentrifuge tube (low protein binding microcentrifuge tubes are no longer necessary) and store them on ice. Discard the pellet. &#160;</li></ol></li><li>Immunoprecipitation (IP)<ol style='list-style-type:decimal;'><li>Mix Protein A and Protein G magnetic beads in a 1:1 ratio. For every IP, prepare 25 &#181;L of beads.</li><li>Wash the beads by adding 1 mL of RIPA buffer (10 mM Tris pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% polyethylene glycol octylphenyl ether, 0.1% SDS, 0.1% Na-deoxycholate; stored at 4 &#176;C) supplemented with protease inhibitors at low concentration (1:1000).</li><li>Use a magnet to allow beads to separate from the washing solution and decant the washing solution (approximately 1 min). Perform the wash step twice.</li><li>Resuspend the beads to the original volume using RIPA buffer supplemented with protease inhibitors (1:1000).</li><li>If necessary, dilute the chromatin using RIPA buffer supplemented with protease inhibitors (1:1000) so that each IP has a volume of 100-200 &#181;L. Reserve 10% of the volume used per IP of chromatin for the input. NOTE: For example, if 200 &#181;L are used per IP, 20 &#181;L of diluted chromatin should be reserved for the input. For a total of four IPs, the total chromatin volume should be diluted to 820 &#181;L.</li><li>Prepare the input. Add 100 &#181;L of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) supplemented with proteinase K (0.5 mg/mL) to the input and incubate the input at 55 &#176;C for 1 h.</li><li>Purify the input using a polymerase chain reaction (PCR) purification kit following the manufacturer's instructions and elute in 50 &#181;L of elution buffer provided in the kit.</li><li>Measure the concentration of the input using a microvolume spectrophotometer and record the results in a notebook. Blank with elution buffer from kit. NOTE: In mouse neurospheres, we used approximately 6 &#181;g of DNA for each IP.</li><li>Run input on a 1% gel or load 1 &#181;L on a DNA Bioanalyzer using standard settings. Store the remainder for use as input in downstream applications.</li><li>Add 10 &#181;L of washed protein A &amp; G magnetic beads for every IP and incubate IP for 1 h at 4 &#176;C. NOTE: For example, add 30 &#181;L of protein A &amp; G magnetic beads for three IPs.</li><li>Place the samples on a magnet and allow beads to separate. Divide chromatin by transferring the previously determined amount into a new 1.5 mL tube.</li><li>Add the antibody and wrap the caps with plastic paraffin film to avoid evaporation. Incubate the samples overnight at 4 &#176;C with rotation at 20 rpm. NOTE: Concentration should be determined empirically following manufacturer recommendations.</li><li>The next day, spin the tubes using a mini centrifuge at room temperature, 2000 x g, for 10 s. Then, add 10 &#181;L of beads to each IP and incubate IP for 3 h at 4 &#176;C with rotation at 20 rpm. &#160;</li></ol></li><li>IP Washes and Protein Digestion<ol style='list-style-type:decimal;'><li>Add 150 &#181;L of RIPA buffer supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow the magnetic beads to separate. Use a pipet to remove the supernatant. Repeat this step five times.</li><li>Add 150 &#181;L of lithium chloride (LiCl) buffer (250 mM LiCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% NP-40, 0.5 % Na-deoxycholate; store at 4 &#176;C) supplemented with protease inhibitors at low concentration (1:1000) to each IP and incubate IP at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove the supernatant.</li><li>Add 150 &#181;L of cold Tris-EDTA (TE) (no protease inhibitors) and incubate the sample at 4 &#176;C with rotation at 20 rpm for 5 min. Place the sample on a magnetic stand and allow magnetic beads to separate. Use a pipet to remove the supernatant.</li><li>After the final washing step, resuspend the sample in 100 &#181;L of TE buffer supplemented with proteinase K (0.5 mg/mL) and incubate the sample at 55 &#176;C for 1 h.</li></ol></li></ol>

native chromatin immunoprecipitation of neurosphere cells

<a href='https://app.jove.com/v/57016/native-chromatin-immunoprecipitation-using-murine-brain-tumor'>Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).</a>This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis.

Native Chromatin Immunoprecipitation of Neurosphere Cells

Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres.&#160;J. Vis. Exp.&#160; (2018).This video ...

Take neurosphere cells in a buffer containing detergents, calcium, and protease inhibitors.&#160;The detergent permeabilizes cells, while the inhibitor inactivates proteases to preserve the chromatin-bound target protein.Add nuclease and incubate. The nuclease uses calcium to digest the chromatin.Add an EDTA-containing buffer that chelates calcium, inactivating the nuclease.Add lysis buffer to lyse the cells, releasing the chromatin.Centrifuge and collect the supernatant.Add antibodies specific to the target protein and incubate with rotation. Centrifuge and remove the supernatant.Introduce magnetic beads targeting the antibody and incubate with rotation.Add lysis buffer to separate non-specifically bound DNA.Magnetically capture the beads and remove the supernatant.Add a high-salt buffer to separate non-specifically bound proteins.Magnetically capture the beads and remove the supernatant.Add a stabilization buffer, magnetically capture the beads, and remove the contaminants.Resuspend in a proteinase-K buffer to digest the proteins, releasing the DNA.

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27 Views. Source: Mendez, F. M. et al., Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres. J. Vis. Exp.  (2018). This video demonstrates the isolation of protein-DNA complexes from neurosphere cells derived from a mouse brain tumor using native chromatin immunoprecipitation. It details the steps for chromatin preparation, immunoprecipitation, washing, and protein digestion to release DNA for downstream analysis. 

Video: Native Chromatin Immunoprecipitation of Neurosphere Cells - Concept

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

JoVE Journal

Cancer Research

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Genetics

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis

In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. Lineage-specific transcription factors are considered to play essential roles in cell specification and differentiation during development. ChIP coupled with high-throughput DNA sequencing (ChIP-seq) is widely used to analyze genome-wide binding sites of transcription factors (or its associated complex) to genomic DNA. However, a large number of cells are required for one standard ChIP reaction, which makes it difficult to study the limited number of isolated primary cells or rare cell populations. In order to understand the regulatory mechanism of oligodendrocyte lineage-specific transcription factor Olig2 in acutely purified mouse OPCs, a detailed method using ChIP-seq to identify the genome-wide binding sites of Olig2 (or Olig2 complex) is shown. First, the protocol explains how to purify the platelet-derived growth factor receptor alpha (PDGFR&#945;) positive OPCs from mouse brains. Next, Olig2 antibody mediated ChIP and library construction are performed. The last part describes the bioinformatic software and procedures used for Olig2 ChIP-seq analysis. In summary, this paper reports a method to analyze the genome-wide bindings of transcriptional factor Olig2 in acutely purified brain OPCs.

Here we present a protocol which is designed to analyze the genome-wide binding of the oligodendrocyte transcription factor 2 (Olig2) in acutely purified brain oligodendrocyte precursor cells (OPCs) by performing low-cell chromatin immunoprecipitation (ChIP), library preparation, high-throughput sequencing and bioinformatic data analysis.

In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. ...

Native Chromatin Immunoprecipitation of Neurosphere Cells

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Native Chromatin Immunoprecipitation of Neurosphere Cells

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFRα+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis