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- Mondal, Tanmoy, 1981, et al.
(författare)
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Chromatin RNA Immunoprecipitation (ChRIP).
- 2017
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Ingår i: Methods in molecular biology. - New York, NY : Humana Press. - 1940-6029. - 9781493973798 ; , s. 65-76
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Bokkapitel (refereegranskat)abstract
- Researchers have recently had a growing interest in understanding the functional role of long noncoding RNAs (lncRNAs) in chromatin organization. Accumulated evidence suggests lncRNAs could act as interphase molecules between chromatin and chromatin remodelers to define the epigenetic code. However, it is not clear how lncRNAs target chromatin remodelers to specific chromosomal regions in order to establish a functionally distinct epigenetic state of chromatin. We developed and optimized chromatin RNA immunoprecipitation (ChRIP) technology to characterize the lncRNAs associated with active and inactive chromatin compartments. Use of ChRIP to identify chromatin-bound lncRNA will further improve our knowledge regarding the functional role of lncRNAs in establishing epigenetic modifications of chromatin.
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| 2. |
- Subhash, Santhilal, 1987, et al.
(författare)
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Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients
- 2017
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; Genetics:124
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Tidskriftsartikel (refereegranskat)abstract
- The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.
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