cell line: IMR90 cell type: primary lung fibroblast cell line treatment: 2Gy timepoint: 24h 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.
cell line: IMR90 cell type: primary lung fibroblast cell line treatment: 2Gy timepoint: 24h 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.