|
| Status |
Public on May 15, 2015 |
| Title |
Control_12 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Human blood, Control
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: whole blood
gender: female
age: 44
sample type: immunoprecipitated
|
| Treatment protocol |
Genomic DNA in human blood samples (IRB #AS 14039) divided into three groups: control (unexposed workers; n = 12), short-term exposure (workers exposed to VOCs for less than 10 years; n = 12), and long-term exposure (workers exposed to VOCs for more than 10 years; n = 12) were used after exposure to VOCs
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total genomic DNA(gDNA) was prepared using the EDTA tubes for gDNA isolation (DNeasy Mini Kit; Qiagen) following the manufacturer's recommendations. gDNA yields were assessed using a NanoDrop (Thermo Fisher Scientific, USA), and the qualities of gDNA were confirmed using a agarose gel electrophoresis (2% agarose). gDNA samples were fragmented into 200–300-bp fragments using a sonicator (Sonics & Materials, Inc., USA). 1 μg of fragmented gDNA was immunoprecipitated using a MethylMiner Methylated gDNA Enrichment Kit (Invitrogen, Carlsbad, CA, USA). The fragmented gDNA(reference gDNA) and immunoprecipitated (IP) gDNA were confirmed using polymerase chain reaction (PCR). gDNA samples were amplified using a Whole Genome Amplification 2 Kit (Sigma, St. Louis, MO, USA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3-dUTP (Cy3) labeled reference gDNA and Cyanine-5-dUTP (Cy5) labeled IP gDNA were parpared from total DNA(1ug;each) using SureTag DNA Labeling Kit (Agilent Technologies). The labeled gDNA were purified with purification column and 1x TE buffer including SureTag DNA Labeling Kit (Agilent Technologies) according to the manufacturer's instructions. Dye incorporation and labeled DNA yield were checked with the NanoDrop (Thermo Fisher Scientific, USA).
|
| |
|
| Channel 2 |
| Source name |
Human blood, Control
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: whole blood
gender: female
age: 44
sample type: input
|
| Treatment protocol |
Genomic DNA in human blood samples (IRB #AS 14039) divided into three groups: control (unexposed workers; n = 12), short-term exposure (workers exposed to VOCs for less than 10 years; n = 12), and long-term exposure (workers exposed to VOCs for more than 10 years; n = 12) were used after exposure to VOCs
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total genomic DNA(gDNA) was prepared using the EDTA tubes for gDNA isolation (DNeasy Mini Kit; Qiagen) following the manufacturer's recommendations. gDNA yields were assessed using a NanoDrop (Thermo Fisher Scientific, USA), and the qualities of gDNA were confirmed using a agarose gel electrophoresis (2% agarose). gDNA samples were fragmented into 200–300-bp fragments using a sonicator (Sonics & Materials, Inc., USA). 1 μg of fragmented gDNA was immunoprecipitated using a MethylMiner Methylated gDNA Enrichment Kit (Invitrogen, Carlsbad, CA, USA). The fragmented gDNA(reference gDNA) and immunoprecipitated (IP) gDNA were confirmed using polymerase chain reaction (PCR). gDNA samples were amplified using a Whole Genome Amplification 2 Kit (Sigma, St. Louis, MO, USA).
|
| Label |
Cy5
|
| Label protocol |
Cyanine-3-dUTP (Cy3) labeled reference gDNA and Cyanine-5-dUTP (Cy5) labeled IP gDNA were parpared from total DNA(1ug;each) using SureTag DNA Labeling Kit (Agilent Technologies). The labeled gDNA were purified with purification column and 1x TE buffer including SureTag DNA Labeling Kit (Agilent Technologies) according to the manufacturer's instructions. Dye incorporation and labeled DNA yield were checked with the NanoDrop (Thermo Fisher Scientific, USA).
|
| |
|
| |
| Hybridization protocol |
2ug of Cy3-labeled reference gDNA and Cy5-labelled IP gDNA were mixed to Hybridization Master Mix containing 10x aCGH blocking agent, 2x HI-RPM Hybridization buffer, Deionized Formamide. On completion of creating mixture, the mixture was reactioned for 3 minutes at 95 and immediately transferred for 37 for 30 minutes following the manufacturers’ instructions, and then 25 ul of 2x Hi-RPM Hybridization Buffer was added to the mixture and hybridized to methylation ChIP/CH3 2x400K Custom array (Agilent Technologies) for 40 hours at 67°C in a rotating Agilent hybridization oven at 20rpm. After hybridization, microarrays were washed 5 minute at room temperature with Agilent Oligo aCGH/ChIP-on-chip Wash buffer1 (Agilent) and 1 minute with 37°C Agilent Oligo aCGH/ChIP-on-chip Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent C scanner (Agilent Technologies) using two color scan(red and green) setting the protocol AgilentG3_CGH for G3 microarrays slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel are set to Green PMT and Red PMT, the gain of PMT is set to 100%, each).
|
| Description |
Methylation of control_12
input samples: Cy3; IP samples: Cy5
|
| Data processing |
The scanned images were analyzed with Agilent Feature Extraction Version 9.1 software using aCGH protocols and settings and enrichments were obtained by extracting the Cy5 (methylated DNA) signals over Cy3 (input DNA) signals. We performed lowness normalization and applied the “Pre-defined Peak Shape Detection 2.0” algorithm provided within the CH3 Analytics software package from Agilent Technologies. We optimized the peak-shape model and identified the significant methylation events among the DNA methylation array data using default algorithm parameters (with a score threshold of 2.0) and applying the option to repeat the peak-finding calculation through the analysis. Correlations of methylation events with genes through the results were determined by comparing predicted peak centers to transcription start sites as defined by the “refFlat” table of RefSeq genes for Hg19 provided at the UCSC Genome Browser Database.
|
| |
|
| Submission date |
May 14, 2015 |
| Last update date |
May 15, 2015 |
| Contact name |
jiyoung hong |
| E-mail(s) |
skydaniel3@naver.com
|
| Phone |
82-10-8992-9924
|
| Organization name |
hanyang university
|
| Street address |
Hanyang Univ.,55, Hanyangdaehak-ro, Sangrok-Gu
|
| City |
Ansan |
| ZIP/Postal code |
426-791 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL20163 |
| Series (2) |
| GSE68908 |
Identification of time-dependent biomarkers and effects of exposure to volatile organic compounds using high-throughput analysis (methylation) |
| GSE68909 |
Identification of time-dependent biomarkers and effects of exposure to volatile organic compounds using high-throughput analysis |
|
