Chromatin Immunoprecipitation of Histone Modifications in Beta Cells

ChIP was performed using the EZ-ChIP Kit (Millipore) according to the manufacturer’s protocol. Beta cells were FACS sorted using Ad.ZsGreen as described earlier. Forty-thousand ZsGreen⁺ (beta) and ZsGreen⁻ (non-beta) cells were collected per experiment for each H3K4me3 and H3K27me3 immunoprecipitation, and 130,000 sorted cells were collected for each KDM6A immunoprecipitation. The primer sets were designed in a previous study^{43 (link)}. Immunoprecipitated DNA was quantified using ABI 7500 real-time quantitative PCR detection system (Life Technologies). The following antibodies were used: anti-H3K4me3 (Millipore #17-614), anti-H3K27me3 (Millipore #17-622), anti-KDM6A (Abcam #ab84190). Data are presented as ChIP reads normalized relative to input controls, and fold-enrichment divided by respective IgG. Three separate islets preparation were used for each figure shown.

Free full text: Click here

Wang H., Bender A., Wang P., Karakose E., Inabnet W.B., Libutti S.K., Arnold A., Lambertini L., Stang M., Chen H., Kasai Y., Mahajan M., Kinoshita Y., Fernandez-Ranvier G., Becker T.C., Takane K.K., Walker L.A., Saul S., Chen R., Scott D.K., Ferrer J., Antipin Y., Donovan M., Uzilov A.V., Reva B., Schadt E.E., Losic B., Argmann C, & Stewart A.F. (2017). Insights into beta cell regeneration for diabetes via integration of molecular landscapes in human insulinomas. Nature Communications, 8, 767.

Publication 2017

EZ-ChIP Kit ABI 7500 real-time quantitative PCR detection system anti-H3K4me3 anti-H3K27me3

Antibodies Beta cells Cells Chip H3k4me3 Immunoprecipitation Kdm6a Primer

Corresponding Organization :

Other organizations : Icahn School of Medicine at Mount Sinai, The Graduate Center, CUNY, Rutgers, The State University of New Jersey, University of Connecticut, Duke University, University of Alabama at Birmingham, New York Genome Center, Imperial College London

Top 5 similar protocols

Variable analysis

independent variables

Cell type (beta cells vs. non-beta cells)

dependent variables

Enrichment of H3K4me3 histone modification
Enrichment of H3K27me3 histone modification
Enrichment of KDM6A protein

control variables

Antibodies used for ChIP (anti-H3K4me3, anti-H3K27me3, anti-KDM6A)
ChIP protocol (EZ-ChIP Kit, Millipore)
Quantification method (ABI 7500 real-time quantitative PCR)
Primer sets used for qPCR

controls

Positive control: Not specified
Negative control: Input controls (ChIP reads normalized relative to input controls)
Biological replicates: Three separate islet preparations used for each figure

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!