ChIP assays were performed with EpiTech ChIP OneDay Kit (334471, Qiagen) following the manufacturer’s protocol, with some modifications. Briefly, PBS- and R-2HG-treated NOMO-1 cells (about 5 × 106) were treated with fresh fixing buffer (1% formaldehyde) for 10 min at 37°C to crosslink DNA and proteins. The reaction was terminated by the addition of stop buffer and incubated at room temperature for 5 min. After cell lysis, the cross-linked chromatin was sonicated to an average size of ~5003bp with a Bioruptor Pico Sonication System and was immunoprecipitated with antibodies against 5hmC, H3K9me3, H3K36me3, and IgG. Purified ChIP DNA was amplified by real-time qPCR using specific primers targeting the 5hmC, H3K9me3, and H3K36me3 peaks on FTO.
Epitech chip oneday kit
Manufactured by Qiagen
Sourced in Italy
The EpiTech CHIP OneDay Kit is a laboratory equipment product designed for chromatin immunoprecipitation (ChIP) experiments. It provides a streamlined workflow for the preparation and purification of ChIP samples in a single day.
Automatically generated - may contain errors
7 protocols using epitech chip oneday kit
1
ChIP Assay of Epigenetic Modifications
2
CHIP Assay on Epigenetic Regulators in Prostate Cancer
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CHIP assays were performed in vehicle- or 5-Aza (5 μM)–treated PCa cells (PC3 or DU145) (4 × 106) and siControl- or siDNMT1-treated PCa cells (PC3 or DU145) (4 × 106) by following the directions of the manufacturer (cat. 334471, EpiTech CHIP OneDay Kit, Qiagen). PCa cells were treated with 1% formaldehyde to cross-link histones to DNA. The cross-linking was stopped by treating the samples with stop buffer for 5 minutes. The chromatin was extracted and fragmented by sonication, and the lysate was used to immunoprecipitation using Protein A + G beads and the following antibodies; H3K9me3 (cat. GAH-6204, Qiagen), H3K27me3 (cat. GAH-9205, Qiagen), or rabbit control IgG (cat. GAH-9205, Qiagen). Immunocomplexes were pulled down and washed, and DNA was isolated to run SYBR qPCR with Zeb2 (assay tile: −0.4 kb; assay position: −3502) (cat. GPH1021580(−)04A, Qiagen) and KLF4 (assay tile: −0.4Kb; assay position: −3110) (cat. GPH1026871(−)04A, Qiagen) specific primers on promoter sites.
3
Chromatin Fragmentation for Protein-DNA Binding
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Protein-DNA Binding Assay was performed using EPItech ChIP One day kit (Qiagen, Milan, Italy) following manufacturing protocol. For each assay, chromatin from about 3 × 106 cells was fragmented to an average size from about 500 to 1500 bp by eight rounds of sonication (Power: 0.5 W, Time: 2 s on, 15 s off; total time 16 s) in 2 mL tubes using the Sonicator 3000 (MISONIX, Part # 3000) (QSonica, LLC, Newtown, CT, USA). The nuclear/DNA fraction was used to analyze the presence of TRPML-1 by western blot analysis.
4
GR Enrichment Profiling by ChIP-qPCR
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The EpiTech ChIP OneDay kit (Qiagen) was employed. IP antibodies used were: Rabbit-IgG (Qiagen), anti-GR (cell signaling) and anti-H3K4me1 (abcam). qRT-PCR was performed by syber green enzyme with the following primers: PANK3-GRE, GAPDH positive control and MYOD1 negative control (Qiagen). GR-GRE primers were planned as follow: Fw – 5′ ATTCTT GTGCCTATGCAGACATTT 3′ and 5′ TGAATGCGTG CATATTCACACTA 3′. % Enrichment was calculated according to the formula: 2^(CT Mean(Input) - CT Mean of (Interested fraction))*100.
5
Quantifying NRF1 Binding to CXCR4
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Chromatin immunoprecipitation assays (ChIP) were carried out with Epitech Chip qPCR primer assay (Qiagen, Germantown, MD, USA). The MCF10A cells of vector, NRF1+, NRF1−(dominant negative for NRF1) were treated with or without E2 (100 pg/mL), for 24 h and analyzed by ChIP assay using the anti-NRF-1 antibody. The CXCR4 promoter region (−109 bp to −98 bp) in the NRF1 precipitated chromatin was amplified by real-time PCR using Epitech Chip qPCR primer assay for human CXCR4 NM_001008540.1 (-)03Kb Cat # GPH1021572(-)03A and Epitech chip one day kit according to the manufacturer’s (Qiagen Science, Inc.) instructions. Chromatin immunoprecipitation qPCR results were calculated using the ΔΔ Ct method.
6
ChIP-seq Profiling Using EpiTech Kit
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Epigenomic Analysis of NDRG1 Promoter in PCa
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