Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Mendez, Flor; Núñez, Felipe J.; Zorrilla-Veloz, Rocío I.; Löwenstein, Pedro R.

doi:10.3791/57016

Cited by 4 publications

(3 citation statements)

References 13 publications

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“…1 IRMs are any substance that binds to the antibody or beads used in the assay; background (noise) occurs when the intended bio-molecular target ( 2 BMT) is not the only 1 IRM in the immunoprecipitation step. Any nucleic acid chain that is in a stable complex with immuno-reactive molecules is isolated from other components through an immunoprecipitation reaction using an antibody bound to magnetic or agarose beads [ [3] , [4] , [5] , [6] , [7] , [8] ]. Once this DNA is isolated, it can be analyzed by microarray, qPCR, or sequencing.…”

Section: Introductionmentioning

confidence: 99%

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A novel method for the normalization of ChIP-qPCR data

Solomon

Allan

2021

MethodsX

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ChIP-qPCR permits the study of protein and chromatin interactions. The general technique can apply to the study of the interactions of protein with RNA, and the methylation state of genomic DNA. While the technique is vital to our understanding of epigenetic processes, there is much confusion around the proper normalization methods. Percent Input has recently emerged as a normalization standard, due to its reproducibility and accuracy. This method relies on the use of a constant volume of ChIP Isolate in each qPCR assay. Researchers may accidentally run qPCR assays with a constant amount of isolate, a common practice for RT-qPCR; however, the traditional Percent Input method cannot accurately normalize these data. We developed a novel method that can normalize these data to provide the same reproducible Percent Input value. Here, we present evidence that this novel method of normalizing ChIP-qPCR data works with real samples. Later, we present a mathematical proof which shows how a Percent Input value calculated from Cq (quantification cycle) values obtained from qPCR run with a constant amount (in nanograms of DNA in ChIP isolate) is equal to the traditional Percent Input calculated from quantification cycle (Cq) values obtained from running a constant volume of ChIP isolate. Increases the number of possible data points per sample End values are the same % Input values as the traditional normalization method

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Section: Introductionmentioning

confidence: 99%

“…Additional controls require a thorough understanding of the target that is not always available to the researcher when designing a ChIP, RIP, or meDIP protocol [ 3 , 9 ]. These involve designing primers that target a region known to associate or not associate with the immuno-reactive molecule of interest.…”

Section: Introductionmentioning

confidence: 99%

A novel method for the normalization of ChIP-qPCR data

Solomon

Allan

2021

MethodsX

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“…The primer sequences are listed in Table 2. The relative enrichment was calculated as previously described [23]. Two biological replicates were performed for each histone mark, which gave similar results.…”

Section: Test Of the Pull-down Efficiency Of N-chip By Qrt-pcr Assaysmentioning

confidence: 99%

A native chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in strawberry fruits

et al. 2020

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Background: Covalent modifications of histones and histone variants have great influence on chromatin structure, which is involved in the transcriptional regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful tool for studying in vivo DNA-histone interactions. Strawberry is a model for Rosaceae and non-climacteric fruits, in which histone modifications have been implicated to affect fruit development and ripening. However, a validated ChIP method has not been reported in strawberry, probably due to its high levels of polysaccharides which affect the quality of prepared chromatin and the efficiency of immunoprecipitation. Results: We describe a native chromatin immunoprecipitation (N-ChIP) protocol suitable for strawberry by optimizing the parameters for nuclei isolation, chromatin extraction, DNA fragmentation and validation analysis using quantitative real-time PCR (qRT-PCR). The qRT-PCR results show that both the active mark H3K36me3 and the silent mark H3K9me2 are efficiently immunoprecipitated for the enriched regions. Compared to X-ChIP (cross-linked chromatin followed by immunoprecipitation), our optimized N-ChIP procedure has a higher signal-to-noise ratio and a lower background for both the active and the silent histone modifications. Furthermore, high-throughput sequencing following N-ChIP demonstrates that nearly 90% of the enriched H3K9/K14ac peaks are overlapped between biological replicates, indicating its remarkable consistency and reproducibility. Conclusions: An N-ChIP method suitable for the fleshy fruit tissues of woodland strawberry Fragaria vesca is described in this study. The efficiency and reproducibility of our optimized N-ChIP protocol are validated by both qRT-PCR and high-throughput sequencing. We conclude that N-ChIP is a more suitable method for strawberry fruit tissues relative to X-ChIP, which could be combined with high-throughput sequencing to investigate the impact of histone modifications in strawberry and potentially in other fruits with high content of polysaccharides.

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Chromatin Immunoprecipitation Assays on Medulloblastoma Cell Line DAOY

Dobson¹,

Swaminathan²

2022

Methods in Molecular Biology

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Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Cited by 4 publications

References 13 publications

A novel method for the normalization of ChIP-qPCR data

A novel method for the normalization of ChIP-qPCR data

A native chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in strawberry fruits

Chromatin Immunoprecipitation Assays on Medulloblastoma Cell Line DAOY

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