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Sample GSM2590761 Query DataSets for GSM2590761
Status Public on Mar 31, 2018
Title GM12878_4Gy_48h
Sample type genomic
 
Channel 1
Source name GM12878, 4Gy, 48h, methylated
Organism Homo sapiens
Characteristics cell line: GM12878
cell type: lymphoblastoid cell line
treatment: 4Gy
timepoint: 48h
sample type: methylated DNA
Growth protocol Human lymphoblastoid cells (GM12878, suspension cells) and primary human lung fibroblasts (IMR-90, adherent cells) were obtained from Corriell Institute for medical Research (Camden, New Jersey, USA). Cells were cultivated at 37°C / 5% CO2 in RPMI-1640 2mM L-glutamine and Eagle's Minimum Essential Medium, respectively.
For each timepoint and dose, 10 million cells were irradiated with an x-ray beam at room temperature with 240 kV tube current (YXLON Maxishot; Hamburg, Germany) filtered with 3 mm beryllium. Absorbed dose was measured with a PTW Unidos dosimeter (PTW Freiburg GmbH, Germany). Dose rate was 1 Gy/min at 13 mA. No fresh medium was added to cells after irradiation. Sham irradiated samples were treated the very same way, apart from not being irradiated (e.g. taken out of the incubator for the same time span, brought to the same room).
Extracted molecule genomic DNA
Extraction protocol DNA was randomly sheared using the Covaris M220 according to the manufacturers’ protocol (Covaris protocol truChIP™ Chromatin Shearing Reagent Kit, PN 010179, Rev C 15 August 2013) with the following parameters: average incident power= 10 W; Peak Incident power= 50 W; Duty factor= 20 %; Cycles/Burst= 200; Duration=80 s. The optimal fragment length of 300-500 bp was verified by gel electrophoreses. The methylated portion of the genome was enriched by immunoprecipitation employing the MagMeDIP kit (Magnetic Methylated DNA Immunoprecipitation kit, Diagenode, Version 1 08.10). In brief, for each sample, 1 µg of sheared DNA was denatured at 95°C for 3 min in a thermocycler. After denaturation, samples were immediately put on ice for 10 min and 5% of each sample were removed for later use as an input control. Immunoprecipitation with monoclonal antibody against 5-methyl cytidine and magnetic beads was done overnight at 4°C on a rotating wheel. The next day, magnetic beads with bound complexes of antibody and methylated DNA were washed for three times and DNA was detached from the beads with proteinase K. Immunoprecipitated DNA as well as input control DNA were purified using the QiaQuick PCR-purification kit (QIAGEN®, QIAquick®). Amplification of DNA was performed with the GenomePlex® Complete Whole Genome Amplification (WGA) Kit according to the protocol (Sigma-Aldrich; Version WGA2, 02/12-1) apart from omitting the fragmentation step.
Label Cy3
Label protocol Samples were labeled with Cy3 and reference DNA (GM12878) was labeled with Cy5 according to manufacturer's protocol (Agilent® Oligonucleotide Array-Based CGH for Genomic DNA Analysis Enzymatic Labeling for Blood, Cells, or Tissues, ProtocolVersion 7.3 March 2014). Dye swaps were also performed.
 
Channel 2
Source name GM12878, 4Gy, 48h, input
Organism Homo sapiens
Characteristics cell line: GM12878
cell type: lymphoblastoid cell line
treatment: 4Gy
timepoint: 48h
sample type: matching control gDNA
Growth protocol Human lymphoblastoid cells (GM12878, suspension cells) and primary human lung fibroblasts (IMR-90, adherent cells) were obtained from Corriell Institute for medical Research (Camden, New Jersey, USA). Cells were cultivated at 37°C / 5% CO2 in RPMI-1640 2mM L-glutamine and Eagle's Minimum Essential Medium, respectively.
For each timepoint and dose, 10 million cells were irradiated with an x-ray beam at room temperature with 240 kV tube current (YXLON Maxishot; Hamburg, Germany) filtered with 3 mm beryllium. Absorbed dose was measured with a PTW Unidos dosimeter (PTW Freiburg GmbH, Germany). Dose rate was 1 Gy/min at 13 mA. No fresh medium was added to cells after irradiation. Sham irradiated samples were treated the very same way, apart from not being irradiated (e.g. taken out of the incubator for the same time span, brought to the same room).
Extracted molecule genomic DNA
Extraction protocol DNA was randomly sheared using the Covaris M220 according to the manufacturers’ protocol (Covaris protocol truChIP™ Chromatin Shearing Reagent Kit, PN 010179, Rev C 15 August 2013) with the following parameters: average incident power= 10 W; Peak Incident power= 50 W; Duty factor= 20 %; Cycles/Burst= 200; Duration=80 s. The optimal fragment length of 300-500 bp was verified by gel electrophoreses. The methylated portion of the genome was enriched by immunoprecipitation employing the MagMeDIP kit (Magnetic Methylated DNA Immunoprecipitation kit, Diagenode, Version 1 08.10). In brief, for each sample, 1 µg of sheared DNA was denatured at 95°C for 3 min in a thermocycler. After denaturation, samples were immediately put on ice for 10 min and 5% of each sample were removed for later use as an input control. Immunoprecipitation with monoclonal antibody against 5-methyl cytidine and magnetic beads was done overnight at 4°C on a rotating wheel. The next day, magnetic beads with bound complexes of antibody and methylated DNA were washed for three times and DNA was detached from the beads with proteinase K. Immunoprecipitated DNA as well as input control DNA were purified using the QiaQuick PCR-purification kit (QIAGEN®, QIAquick®). Amplification of DNA was performed with the GenomePlex® Complete Whole Genome Amplification (WGA) Kit according to the protocol (Sigma-Aldrich; Version WGA2, 02/12-1) apart from omitting the fragmentation step.
Label Cy5
Label protocol Samples were labeled with Cy3 and reference DNA (GM12878) was labeled with Cy5 according to manufacturer's protocol (Agilent® Oligonucleotide Array-Based CGH for Genomic DNA Analysis Enzymatic Labeling for Blood, Cells, or Tissues, ProtocolVersion 7.3 March 2014). Dye swaps were also performed.
 
 
Hybridization protocol Samples were hybridized on a whole genome array (Agilent®, Santa Clara, CA, USA; SurePrint G3 Human Genome CGH Microarray 2x400K, 5.3 kb spacing).
Scan protocol Agilent® Oligonucleotide Array-Based CGH for Genomic DNA Analysis, Version 7.3 March 2014; Agilent® G2505C; Scan Control Version A.8.1.3; resolution 3 µm; 16 bit TIFF; no XDR; Images were quantified using Agilent Feature Extraction Software (version 12.0).
Description meDIP
Data processing Agilent Feature Extraction Software (v 12.0) was used for background subtraction and LOWESS normalization.
 
Submission date Apr 27, 2017
Last update date Feb 12, 2019
Contact name Benjamin Becker
E-mail(s) benjamin3becker@bundeswehr.org
Organization name Bundeswehr Institute for Radiobiology
Department Genomics II
Street address Neuherbergstr. 11
City Munich
ZIP/Postal code 80804
Country Germany
 
Platform ID GPL9777
Series (2)
GSE98307 Regional preferences of DNA methylation changes in response to X-ray-irradiation [Agilent-021850 array]
GSE98308 Regional preferences of DNA methylation changes in response to X-ray-irradiation

Data table header descriptions
ID_REF
VALUE Normalized log2 ratio (Cy3/Cy5)

Data table
ID_REF VALUE
1 -3.63E-01
2 -8.69E-01
3 -7.61E-01
4 2.75E-01
5 -2.73E-01
6 -4.28E-01
7 2.79E-01
8 -3.94E-03
9 3.69E-01
10 -3.64E-01
11 1.35E-01
12 -4.46E-01
13 -1.31E-01
14 -1.93E-01
15 -2.99E-02
16 -2.03E-01
17 2.63E-01
18 -1.54E-01
19 5.05E-01
20 -4.19E-01

Total number of rows: 420288

Table truncated, full table size 6659 Kbytes.




Supplementary file Size Download File type/resource
GSM2590761_12878_meDIP_4Gy_48h.txt.gz 44.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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