ChIP was performed using the EZ-ChIP Kit (#Magna0001, Millipore) according to manufacturer’s protocol as described previously (Wang et al., 2017 ). Whole human cadaveric islets were dispersed as described previously. A minimum of three separate islet preparations were used for each figure shown. 2×106 cells were collected per experiment for each SMAD2/3, SMAD4, KDM6A and MEN1 immunoprecipitation. Immunoprecipitated DNA was quantified using ABI 7500 real-time quantitative PCR detection system (Life Technologies). Data are presented as binding signals calculated by normalizing the ChIP signals relative to input controls and subsequently subtracting the IgG value from the respective antibody. The resulting values below zero indicated no binding and depicted as ‘zero’ in ChIP plots. Error bars indicate mean ± SEM. The primer sets for CDKN1A and CDKN1C were described previously (Koinuma et al., 2009 (link); Wang et al., 2017 ; Yang et al., 2009 (link)). The antibodies and the primer sequences used are described in the Key Resources Table .
Abi 7500 real time quantitative pcr detection system
Manufactured by Thermo Fisher Scientific
The ABI 7500 real-time quantitative PCR detection system is a laboratory instrument designed for high-performance real-time PCR analysis. It provides precise and sensitive detection and quantification of DNA or RNA targets.
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3 protocols using abi 7500 real time quantitative pcr detection system
1
ChIP-qPCR Protocol for Islet Epigenetics
2
Chromatin Immunoprecipitation of Histone Modifications in Beta Cells
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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 study43 (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.
3
Quantitative ChIP Assay for PDX1
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ChIP was performed using the EZ-ChIP Kit (Millipore) according to the manufacturer’s protocol. Three batches of cadaveric human islets (250–300 IEQs) were used per experiment for each PDX1 immunoprecipitation. Insulinoma tissue was chopped into 2-3mm3 pieces and disassociated. Cells were counted using a hemocytometer. Chromatin was prepped from 250,000–300,000 cells according to the EZ-ChIP Kit protocol. The primer sets were designed based on the hypermethylated CpGs in insulinomas and PDX1 ChIP-seq peaks in whole human islets reported by Pasquali et al.16 (link) and shown in Supplementary Fig. 10 (see Supplementary Data 14 for primer sequences). Immunoprecipitated DNA was quantified using ABI 7500 real-time quantitative PCR detection system (Life Technologies). The anti-PDX1 antiserum (AB2027) was kindly provided by Prof. Christopher Wright at Vanderbilt University and was diluted 1:100 for each ChIP experiment. Data are presented as fold-enrichment of the ChIP signal over the IgG signal.
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