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| Status |
Public on Jan 22, 2013 |
| Title |
Small lung tumor tissue with Dnmt3a knocked out |
| Sample type |
SRA |
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| Source name |
lung tumors
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| Organism |
Mus musculus |
| Characteristics |
genotype/variation: KOS
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| Treatment protocol |
adenoviral Cre recombinase was delivered to mice of 6-12 weeks old via intratracheal or intranosal approach
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| Growth protocol |
K-ras LSL-G12D mice were crossed with the Dnmt3a conditional KO mice. After adenoviral Cre recombinase –mediated recombination, mice were generated with lung cells that carried the K-ras G12D mutation and were homozygous (KO) and WT at the Dnmt3a locus. Tumors were flash-frozen in liquid nitrogen and stored at -80°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5 µg of high molecular weight DNA were used for fragmentation using the Covaris S2 AFA System in a total volume of 100 µl. Fragmentation-run parameters: Duty cycle 10%; Intensity: 5; Cycles/burst: 200; Time: 3 min; number of cycles: 3, resulting in a total fragmentation-time of 180 s. Fragmentation was confirmed with a 2100 Bioanalyzer (Agilent Technologies) using a DNA1000 chip. The fragmented DNAs were concentrated to a final volume of 75 µl using a DNA Speed Vac. End repair of fragmented DNA was carried out in a total volume of 100 µl using the Paired End DNA Sample Prep Kit (Illumina) as recommended by the manufacturer. For the ligation of the adaptors, the Illumina Early Access Methylation Adaptor Oligo Kit and the Paired End DNA Sample Prep Kit (Illumina) were used, as recommended by the manufacturer. For the size selection of the adaptor-ligated fragments, we used the E-Gel Electrophoresis System (Invitrogen) and a Size Select 2% precast agarose gel (Invitrogen). Each fragmented DNA was loaded on two lanes of the E-gel. Electrophoresis was carried out using the “Size Select” program for 16 min. According to the standard loaded (50 bp DNA Ladder, Invitrogen), 240 bp fragments were extracted from the gel, pooled, and directly transferred to bisulfite treatment without further purification. For the bisulfite treatment we used the EZ-DNA Methylation Kit (Zymo) as recommended by the manufacturer with the exception of a modified thermal profile for the bisulfite conversion reaction. The conversion was carried out in a thermal cycler using the following thermal profile: 95°C for 15 s, 50°C for 1 h, repeat from step 1, 15×, 4°C for at least 10 min. The libraries were subsequently amplified, using the Fast Start High Fidelity PCR System (Roche) with buffer 2, and Illuminas PE1.1 and PE2.1 amplification primers. PCR thermal profile: 95°C for 2 min, 95°C for 30 s, 65°C for 20 s, 72°C for 30 s, then repeat from step 2, 11×, 72°C for 7min, hold at 4°C. PCR reactions were purified on PCR purification columns (MinElute, Qiagen) and eluted in 20 µl elution buffer (Qiagen).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Casava1.8.1 software used for basecalling.
Preprocessing, read trimming: shore 0.6.2
Preprocessing, quality filtering: shore 0.6.2
BS-mapping: bsmap 2.0
computation of methylation ratios: methratio.py (part of bsmap package)
Genome_build: mm9
Supplementary_files_format_and_content: Bedgraph-format containing the genomic methylation ratios of the respective sample
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| Submission date |
Jun 12, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Guenter Raddatz |
| Organization name |
German Center for Cancer Research
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| Street address |
Im Neuenheimer Feld 580
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| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE38651 |
Dnmt3a protects active chromosome domains against cancer-associated hypomethylation |
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| Relations |
| SRA |
SRX153156 |
| BioSample |
SAMN01047959 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM946857_kos.bedgraph.gz |
198.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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