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Genomic Informatics in the Healthcare System
Published in Salvatore Volpe, Health Informatics, 2022
Another possible explanation for the phenotypic heterogeneity among cases is epigenetic differences. Epigenetic processes such as DNA methylation, histone modifications, chromatin remodeling, and non-coding RNAs can alter the activity of a gene without changing the DNA sequence. To further understand and identify the detail of the heterogeneity, a strategy such as the chromatin immunoprecipitation sequencing (ChIP-Seq) or RNA-Seq can be used. Exploring factors that can account for the phenotypic variability may provide insight into the pathways involved in disease. Furthermore, the comprehensive genetic evaluation and investigation of various individuals with these conditions will likely enlighten the fields of genomic locus heterogeneity and allelic heterogeneity of the genes with a broad spectrum of detected variants.
ChIP-seq analysis
Published in Altuna Akalin, Computational Genomics with R, 2020
ChIP-seq stands for chromatin immunoprecipitation followed by sequencing, and is an experimental method for finding locations on DNA which are bound by proteins. It has been extensively used to study in-vivo binding preferences of transcription factors, and genomic distribution of modified histones.
The Role of Epigenetics in Breast Cancer: Implications for Diagnosis, Prognosis, and Treatment
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
Amy M. Dworkin, Tim H.-M. Huang, Amanda E. Toland
Commonly used methods for detecting histone modifications usually rely on chromatin immunoprecipitation (ChIP). In ChiP, DNA is cross-linked to chromatin, followed by co-immunoprecipitation using antibodies against specific histone residues or chromatin proteins. “ChIP” DNA can be used in microarrays, cloning, and sequencing (11). Techniques for detecting histone modifications have not yet been adapted for clinical application.
The gut microbiota mediates protective immunity against tuberculosis via modulation of lncRNA
Published in Gut Microbes, 2022
Fang Yang, Yi Yang, Lingming Chen, Zhiyi Zhang, Linna Liu, Chunmin Zhang, Qiongdan Mai, Yiwei Chen, Zixu Chen, Tao Lin, Liang Chen, Huixin Guo, Lin Zhou, Hongbo Shen, Xinchun Chen, Lei Liu, Guoliang Zhang, Hongying Liao, Lingchan Zeng, Gucheng Zeng
Chromatin Immunoprecipitation and ChIP-qPCR: 1 × 106 cells were prepared for each round of ChIP. ChIP was performed using EZ MAGNA ChIP assay kit (millipore) according to the manufacturer’s manual. Briefly, after fixation of cells with formaldehyde (1%), Chromatin DNA was immunoprecipitated with the following antibodies: rabbit anti-H3K9Me1 (ab9045, Abcam), mouse anti-H3K27Me3 (ab6002, Abcam), rabbit anti-H3K4Me3 (ab8580, Abcam), rabbit anti-H3 (ab1791, Abcam), rabbit anti-H3K27Me2 (ab24684, Abcam), mouse anti-H3k9Me2 (ab1220, Abcam), rabbit anti-H3K9Me3 (ab8898, Abcam). PCRs were performed in 20-μl reaction mixtures containing 2 μl of DNA, 10 μl of SYBR green master mix and 1 μl of each primer. The following gene-specific ChIP-qPCR primers were used: The PCR amplimers were confirmed to be a single band of the correct size by agarose gel electrophoresis. ChIP-qPCR data were normalized by the % input method. Results are expressed as means ± SEM of the indicated number of independent determinations.
Precise nanoinjection delivery of plasmid DNA into a single fibroblast for direct conversion of astrocyte
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Hang-Soo Park, Hyosung Kwon, Jewon Yu, Yeonju Bae, Jae-Yong Park, Kyung-Ah Choi, Yeonho Choi, Sunghoi Hong
Chromatin immunoprecipitation (ChIP) assays were preformed according to the EZ-ChIP™ kit (Millipore, Billerica, MA) manufacturer’s protocol. MEF cells were plated in 100-mm plates and cultured for 1 day and then transduced with a retroviral vector expressing the Sox2 gene. Three days after transduction, approximately 3 × 106 cells were cross-linked with 1% formaldehyde for 10 min and harvested in the presence of protease inhibitor cocktail (Millipore, Billerica, MA). An EZ-ChIP™ kit (Milipore, Billerica, MA) and a provided protocol were used for cell lysis and immunoprecipitation process. Sox2 antibody (Millipore, Billerica, MA) was used to precipitate Sox2 protein–DNA complexes. After precipitation, the DNA was reversed from protein–DNA complexes by treatment with NaCl and then recovered by phenol extraction. PCR was performed to detect specifically bound DNA. Used primer sequence is listed in Supplementary Table 2.
Suppressive effects of metformin on T-helper 1-related chemokines expression in the human monocytic leukemia cell line THP-1
Published in Endocrine Research, 2018
Yen-Chun Chen, Chang-Hung Kuo, Ying-Ming Tsai, Yi-Ching Lin, Hui-Pin Hsiao, Bai-Hsiun Chen, Yi-Ting Chen, Shih-Ling Wang, Chih-Hsing Hung
Chromatin immunoprecipitation assay was performed following the study of Lee et al.22 and according to the manufacturer’s instructions. THP-1 cells (1 × 106/mL) were pretreated without or with metformin (1 and 20 μM) for 2 hours and stimulated with LPS (0.2 µg/mL) for 1 hour. The chromatin was immunoprecipitated with antibodies for the acetylated histones H3 and H4 (Upstate Biotechnology, Waltham, MA, USA). Sequences of primers for the polymerase chain reaction in the proximal promoter region of IP-10 were as follows: 5′-GAGGGAAATTCCGTAACTTGG-3′ (sense) and 5′-TCAGAAAACGTGGGGCTAGT-3′ (antisense), encompassing the subregions (+9/−172) with two NF-κB binding sites.23 All experiments were performed in triplicate.