Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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OBFOXO1CHIPSeq

ABSTRACT: This study is designed to identify gene promoters and enhancers target of foxo1, an transcription factor activated by melatonin in osteoblast cells. Protocol:1. Primary osteoblast cells were grown at 70-80% confluency in 150mm culture dishes.2. These cells were then treated with melatonin for 24h under serum-deprived condition.3. After the completion of treatment cells were fixed with medium containing formaldehyde, washed with PBS and reaction was stopped by adding glycine stop solution. Cells were collected in cell scraping solution. 4. Further sonication and CHIP reaction with foxo1 antibody was performed using Active Motif CHIP-IT express kit following manufacturers instructions.5. After CHIP DNA was purified using Active Motif Chromatin IP DNA purification Kit following manufacturers instructions. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER:

PROVIDER: E-ERAD-105 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This study focuses on epigenetic reprogramming in the mouse germ line: DNA methylation marks, including those of imprinted genes, are thought to be erased between E11.5 and E13.5 in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. DNA methylation patterns are then re-established during the de novo methylation phase in the male germ line several days later (around E15.5) and in the female germ line after birth during adult life. Epigenetic reprogramming in PGCs is poorly understood mainly because of the technical challenges that arise from very low cell numbers in the embryo. The aim of this study is to create genome-wide maps of DNA methylation patterns of male and female PGCs at crucial time points during epigenetic reprogramming and to investigate the changes in those profiles on a single-gene level. We have already successfully prepared and sequenced the first set of BS-Seq libraries of PGCs with Illumina's GAIIx platform. From this, we gained significant insight into the global DNA methylation levels and methylation patterns over multy-copy-loci such as repeat elements. In order further enhance our analysis and finish this project for publication, it is crucial to increase the genomic coverage of our datasets. This will also allow us to link the BS-Seq data with other MeDIP-Seq and RNA-Seq datasets that have already been created in this study.Protocol: Input DNA was sonicated using a Bioruptor UCD-200 (Diagenode) to a final size distribution of 300bp 1000bp. End-repair and A-tailing were performed with the NEBNext DNA Sample Prep Master Mix Set 1. Illuminas Early Access Methylation Adaptor Oligo Kit was used for the adapter ligation. The adapter-ligated DNA was treated with sodium-bisulfite using the Imprint DNA Modification Kit from Sigma-Aldrich according to the manufacturers instructions for the two-step protocol. After the clean up, the bisulfite-treated DNA was amplified using PfuTurbo Cx Hotstart DNA Polymerase from Agilent with 18 cycles of amplification. The entire PCR reaction was run on a 1.5% agarose TBE gel and size selection was performed for DNA fragments between 200bp 250bp. DNA from the excised gel piece was purified with the Qiagen Gel Extraction Kit.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

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OBFOXO1CHIPSeq

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