• Title/Summary/Keyword: Chromatin

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ChIP-seq Library Preparation and NGS Data Analysis Using the Galaxy Platform (ChIP-seq 라이브러리 제작 및 Galaxy 플랫폼을 이용한 NGS 데이터 분석)

  • Kang, Yujin;Kang, Jin;Kim, Yea Woon;Kim, AeRi
    • Journal of Life Science
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    • v.31 no.4
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    • pp.410-417
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    • 2021
  • Next-generation sequencing (NGS) is a high-throughput technique for sequencing large numbers of DNA fragments that are prepared from a genome. This sequencing technique has been used to elucidate whole genome sequences of living organisms and to analyze complementary DNA (cDNA) or chromatin immunoprecipitated DNA (ChIPed DNA) at the genome level. After NGS, the use of proper tools is important for processing and analyzing data with reasonable parameters. However, handling large-scale sequencing data and programing for data analysis can be difficult. The Galaxy platform, a public web service system, provides many different tools for NGS data analysis, and it allows researchers to analyze their data on a web browser with no deep knowledge about bioinformatics and/or programing. In this study, we explain the procedure for preparing chromatin immunoprecipitation-sequencing (ChIP-seq) libraries and steps for analyzing ChIP-seq data using the Galaxy platform. The data analysis steps include the NGS data upload to Galaxy, quality check of the NGS data, premapping processes, read mapping, the post-mapping process, peak-calling and visualization by window view, heatmaps, average profile, and correlation analysis. Analysis of our histone H3K4me1 ChIP-seq data in K562 cells shows that it correlates with public data. Thus, NGS data analysis using the Galaxy platform can provide an easy approach to bioinformatics.

Identification of hRad21-Binding Sites in Human Chromosome

  • Chin Chur;Chung Byung-Seon
    • Genomics & Informatics
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    • v.4 no.1
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    • pp.11-15
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    • 2006
  • The aim of this study is to identify hRad21-binding sites in human chromosome, the core component of cohesin complex that held sister chromatids together. After chromatin immunoprecipitation with an hRad21 antibody, it was cloned the recovered DNA and sequenced 30 independent clones. Among them, 20 clones (67%) contained repetitive elements including short interspersed transposable elements (SINE or Alu elements), long terminal repeat (LTR) and long interspersed transposable elements (LINE), fourteen of these twenty (70%) repeats clones had Alu elements, which could be categorized as the old and the young Alu Subfamily, eleven of the fourteen (73%) Alu elements belonged to the old Alu Subfamily, and only three Alu elements were categorized as young Alu subfamily. There is no CpG island within these selected clones. Association of hRad21 with Alu was confirmed by chromatin immunoprecipitation-PCR using conserved Alu primers. The primers were designed in the flanking region of Alu, and the specific Alu element was shown in the selected clone. From these experiments, it was demonstrated that hRad21 could bind to SINE, LTRs, and LINE as well as Alu.

In vitro Culture Conditions for the Mouse Preantral Follicles Isolated by Enzyme Treatment

  • Kim, Dong-Hoon;Seong, Hwan-Hoo;Lee, Ho-Joon
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.4
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    • pp.532-537
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    • 2008
  • In order to investigate the factors affecting the culture of mouse preantral follicles in vitro, we examined the effect of culture media, protein supplements, and culture period on their growth. The oocyte diameter (initial size: $55.6{\pm}2.5{\mu}m$) was progressively increased during culture, and the maximum size ($72.0{\pm}2.4{\mu}m$) was reached on day 10 of the in vitro culture. The chromatin configuration in the germinal vesicle (GV) oocyte progressively shifted from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN). On day 10 of the culture, most of the oocytes progressed to the SN pattern. The survival and metaphase II rates of the oocytes in alpha-minimal essential medium (alpha-MEM) were significantly higher (p<0.05) than those in Waymouth and tissue culture medium (TCM)-199. As a protein source, fetal bovine serum (FBS) was more suitable for the culture of mouse preantral follicles as compared to human follicular fluid (hFF) and bovine serum albumin (BSA); the optimal concentration of FBS was 5%. These results suggest that in a culture of mouse preantral follicles, alpha-MEM and 5% FBS are an optimal medium and a protein source, respectively; further, the 10 days of culture is required for the complete growth of oocytes in this culture system.

Evaluating the Existence of Small Compressed Binucleated Squamous Cells in ASC-H

  • Okodo, Mitsuaki;Okayama, Kaori;Kitamura, Hiroshi;Shiina, Natsuko;Caniz, Timothy;Ono, Midori;Yabusaki, Hiromi
    • Asian Pacific Journal of Cancer Prevention
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    • v.17 no.10
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    • pp.4665-4669
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    • 2016
  • Purpose: To evaluate the legitimacy of a diagnosis of ASC-H in 5 cases which were followed up monthly for over 2 years with both cytology and HPV testing. Methods: Some 5 cases out of a total of 25.0 self-sampled Pap test patients diagnosed as ASC-H provided 119 specimens over 2 years, with HPV-DNA testing perormed using a E6 primer. Results: Cases 1, 2 and 3 showed SIL after the ASC-H diagnosis, while cases 4 and 5 showed and maintained NILM. Cases 1, 2 and 3 were further characterized by small atypical compressed binucleated cells, in which HPV was detected by in situ PCR. Case 4 showed a high N/C ratio in cells in sheets with a mild increase in chromatin. Case 5 demonstrated a high N/C ratio in small cells with no increase in chromatin. Conclusion: The finding of a compressed binucleated cells can define the difference between degenerated endocervical columnar cells and small atypical cells suggestive of HSIL. When small compressed binucleated squamous cells are detected, there may be a chance of continuing HPV infection and undetected SIL.

Functional characterization of a minimal sequence essential for the expression of human TLX2 gene

  • Borghini, Silvia;Bachetti, Tiziana;Fava, Monica;Duca, Marco Di;Ravazzolo, Roberto;Ceccherini, Isabella
    • BMB Reports
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    • v.42 no.12
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    • pp.788-793
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    • 2009
  • TLX2 is an orphan homeodomain transcription factor whose expression is mainly associated with tissues derived from neural crest cells. Recently, we have demonstrated that PHOX2A and PHOX2B are able to enhance the neural cell-type specific expression of human TLX2 by binding distally the 5' -flanking region. In the present work, to deepen into the TLX2 transcription regulation, we have focused on the proximal 5'-flanking region of the gene, mapping the transcription start site and identifying a minimal promoter necessary and sufficient for the basal transcription in cell lines from different origin. Site-directed mutagenesis has allowed to demonstrate that the integrity of this sequence is crucial for gene expression, while electrophoretic mobility shift assays and chromatin immunoprecipitation experiments have revealed that such an activity is dependent on the binding of a PBX factor. Consistent with these findings, such a basal promoter activity has resulted to be enhanced by the previously reported PHOX2-responding sequence.