Chromatin Immunoprecipitation of H3K27Ac and GR

ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif) according to manufacturer’s instructions. A TissueRuptor (QIAGEN) was used for 45 seconds at maximum speed to disrupt liver tissue, followed by Dounce homogenizing for 70 strokes to release cell nuclei. Nuclear sonication was conducted with an EpiShear Probe Sonicator (Active Motif, 3.5-mm probe) for 8 rounds of 2 minutes per sample, at 37% amplitude. 15 μg sheared chromatin was incubated with anti-H3K27Ac antibody (Active Motif, lot 31814008) or an anti-GR antibody cocktail (Proteintech 24050-1-A, lot 00044414, and Cell Signaling Technology, D8H2, lot 2). For GR ChIP, 30 ng spike-in Drosophila melanogaster chromatin (Active Motif) plus an antibody to the Drosophila-specific histone variant H2Av (Active Motif Spike-in Antibody 61686; lot 34216004) (44 (link)) were include in the IP reaction. Antibody was pulled down with magnetic protein G agarose microspheres (ReSyn Biosciences). After elution and de-crosslinking, ChIP DNA was purified, as per the kit instructions.

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Caratti G., Iqbal M., Hunter L., Kim D., Wang P., Vonslow R.M., Begley N., Tetley A.J., Woodburn J.L., Pariollaud M., Maidstone R., Donaldson I.J., Zhang Z., Ince L.M., Kitchen G., Baxter M., Poolman T.M., Daniels D.A., Stirling D.R., Brocker C., Gonzalez F., Loudon A.S., Bechtold D.A., Rattray M., Matthews L.C, & Ray D.W. (2018). REVERBa couples the circadian clock to hepatic glucocorticoid action. The Journal of Clinical Investigation, 128(10), 4454-4471.

Publication 2018

ChIP-IT High Sensitivity kit TissueRuptor EpiShear Probe Sonicator Spike-in Antibody 61686

Agarose Anti antibody Antibody Antibody cocktail Cell nuclei Chip Chromatin Drosophila Drosophila melanogaster Histone Liver Microspheres Protein g Seconds Sensitivity Strokes Tissue

Corresponding Organization :

Other organizations : Manchester Academic Health Science Centre, University of Manchester, National Cancer Institute, Center for Cancer Research, Molecular Discovery (United Kingdom), GlaxoSmithKline (United Kingdom), University of Leeds, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford

Top 5 similar protocols

Variable analysis

independent variables

Tissue disruption method (TissueRuptor for 45 seconds at maximum speed, followed by Dounce homogenization for 70 strokes)
Nuclear sonication (EpiShear Probe Sonicator for 8 rounds of 2 minutes per sample, at 37% amplitude)
Antibody used for ChIP (anti-H3K27Ac antibody or anti-GR antibody cocktail)

dependent variables

Amount of sheared chromatin (15 μg)
Binding of antibodies to target proteins (H3K27Ac or GR)

control variables

Manufacturer's instructions for the ChIP-IT High Sensitivity kit (Active Motif)
Magnetic protein G agarose microspheres (ReSyn Biosciences) for antibody pulldown
Spike-in Drosophila melanogaster chromatin (30 ng) and Drosophila-specific histone variant H2Av antibody for GR ChIP

positive controls

Spike-in Drosophila melanogaster chromatin (30 ng) and Drosophila-specific histone variant H2Av antibody for GR ChIP

negative controls

None explicitly mentioned

Annotations

Based on most similar protocols

Tissue disruption was performed using a TissueRuptor (QIAGEN) for 45 seconds at maximum speed, followed by Dounce homogenizing for 70 strokes to release cell nuclei (Input protocol).

Nuclear sonication was conducted with an EpiShear Probe Sonicator (Active Motif, 3.5-mm probe) for 8 rounds of 2 minutes per sample, at 37% amplitude (Input protocol).

For GR ChIP, 30 ng spike-in Drosophila melanogaster chromatin (Active Motif) plus an antibody to the Drosophila-specific histone variant H2Av (Active Motif Spike-in Antibody 61686; lot 34216004) were included in the IP reaction (Input protocol).

To achieve optimal inter-sample normalization of ChIP efficiency, Drosophila chromatin (Abcam #58083, 100 ng), which contains fly-specific histone variant H2Av, was spiked in and subsequently immunoprecipitated with 0.4 μg anti-H2Av antibody (Active Motif #39715) (Protocol 1).

Sonication was performed using the Bioruptor (Diagenode) to obtain ∼500 bp DNA fragments (Protocol 2).

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