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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
For a genome-wide approach, ChIP-on-chip was the first widespread method in which DNA fragments are analysed via a microarray analysis. However, with rapid development of NGS, ChIP-sequencing has become the more common methodology employed. In this process, DNA fragments are purified from the ChIP protocol detailed above and used for generating DNA sequencing libraries. Nonetheless, both approaches provide information regarding the binding location and amount of binding of the histone modification of choice (e.g. the histone mark used for the antibody immunoprecipitation step) (29). Each of these protocols however is costly, and produces a large amount of data and requires advanced expertise in bioinformatics and data analysis (30). To overcome these limitations, researchers are now able to analyse the purified DNA fragments via quantitative PCR (qPCR), in a far more targeted approach. This protocol relies heavily on the researchers knowing exactly the region in the genome they wish to target, and the ability to design PCR primers to investigate this area. ChIP protocols require large quantities of starting material, are relatively labour-intensive and, for sequencing protocols, require large amounts of sequencing data in order to reliably determine meaningful differences across datasets.
Next-Generation Sequencing (NGS) for Companion Diagnostics (CDx) and Precision Medicine
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
Il-Jin Kim, Mendez Pedro, David Jablons
Second, Ion Torrent is faster than other NGS systems because the expensive and time-consuming optical sensing is removed, which also makes the equipment cheaper than other competitors. After sample library preparation, PGM 314 chip sequencing takes less than 3 h to complete the sequencing run (www.thermofisher.com). This can be a huge advantage for precision medicine and CDx applications where patients with emergent conditions are waiting for results for drug selection.
Application of NanoString technologies in companion diagnostic development
Published in Expert Review of Molecular Diagnostics, 2019
Jennifer Mary Eastel, Ka Wai Lam, Nga Lam Lee, Wing Yan Lok, Andy Hin Fung Tsang, Xiao Meng Pei, Amanda Kit Ching Chan, William Chi Shing Cho, Sze Chuen Cesar Wong
ChIP-string was reported to be applied in systematic analysis to reveal any epigenomic changes in melanoma progression. A strategy using a ChIP-string assay with 96 designed test probes was used to evaluate the key epigenetic features observed in different cell lines. Six selected histone regions were selected for investigation. They were H2BK5ac, H4K5ac, H3K27ac, H3K4me1, H3K4me3, and H3K27me3, which represented promoters, enhancers or polycomb-repressed regions. Results revealed that the loss of histone acetylations and H3K4me3 on regulatory regions were associated with melanoma development. Upon treatment with histone deacetylase inhibitors, restoration of acetylation on deacetylated loci suppressed the aggressive proliferation of tumorigenic cells [29]. There was also a good correlation between the signal generated from ChIP-string and ChIP-sequencing.
Proteomic approaches for cancer epigenetics research
Published in Expert Review of Proteomics, 2019
Dylan M. Marchione, Benjamin A. Garcia, John Wojcik
The first mechanistic study of an oncohistone was published in 2013 [135]. Lewis et al. reported that cultured cells and tissue slices from diffuse intrinsic pontine gliomas (DIPGs) expressing H3.3K27M displayed markedly low levels of histone H3K27me2/3. Accordingly, H3.3K27M expression was sufficient to reduce H3K27me3 levels in vitro and in vivo via potent inhibition of the EZH2 subunit of PRC2. The study also revealed that H3.3K9M and K36M can likewise inhibit their respective SET domain-containing methyltransferases. The major contribution of MS to this study was the measurement of mutant histone levels relative to wild-type in affected cells. A common misconception is that K27 methylation levels are reduced in K27M-expressing cells simply because lysines are replaced by methionines. In fact, the mutant histone typically represents a minority of total histone H3 (ranging from 3 to 17% in the three DIPG lines tested), supporting dominant negative-type inhibition of K27 methylation [135]. This model was also supported by a concomitant study that primarily used western blotting and ChIP-sequencing to assess mutation-induced changes in the global histone methylation landscape [136].
Transcriptional regulation of PEBP1 expression by androgen receptor in mouse testes
Published in Systems Biology in Reproductive Medicine, 2022
Qiong Deng, Zhu Wang, Ye Du, Ying Zhang, Hui Liang
ChIP-sequencing was conducted to detect the expression profiles of Sertoli cells from WT mice (basal condition and testosterone-treated). Sertoli cells were collected following the previously described protocol (Chen et al. 2014; Chang et al. 2008), and ChIP-sequencing was performed as described in the Materials and Methods section. The ChIP samples were further examined, sequenced, and analyzed by Novogene (Beijing, China; Figure 1). Based on the fold enrich value (the ratio of RPM value in IP to rpm value in input), hierarchical clustering analysis is performed in Figure 1A. Rpm is the ratio of 1 m reads in a single sample to the peak. The fold enrich value (the ratio of RPM in IP to RPM in input) of each comparison group in the peak annotation area was calculated. The enrichment level of different IP experiments was displayed by boxplot in Figure 1B. At the same time, we carried out GO enrichment analysis (Figure 1C) and KEGG enrichment analysis (Figure 1D) for genes related to different peaks. GO enrichment showed their genes are enriched in developmental process, metabolic process, regulation of signaling, and protein binding. In Figure 1D, there genes are enriched in signaling pathway and tumorigenesis. Bioinformatics analysis indicated effective androgen-responsive elements (AREs) located in the promotor of Pepb1 gene (ARE: −1516/-1520). Primers were designed and synthesized against the promoter of Pebp1. The ChIP- quantitative polymerase chain reaction (qPCR) data using Pebp1 primers (F: 5ʹ-AGC CTC AAC CAA GAT CCA GC-3ʹ, R: 5ʹ-CCG GAG ATA AGG CCA TGC TT-3ʹ) showed target bands by agarose electrophoresis (Figure 1E).