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| Status |
Public on Feb 13, 2015 |
| Title |
HpaII_1.0T |
| Sample type |
SRA |
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| Source name |
HEK 293T cells, cells treated for three days with 1.0 µM of 5-aza-CdR
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK 293T
cell type: non-malignant cell line
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| Treatment protocol |
HEK 293T cells were treated for three days with 1.0 µM of 5-aza-CdR. Cells with no drug were grown as controls. Tissue culture medium was changed every day for both control and treated cells, to maintain the drug stability during treatment. After collection, treated cells were remained in culture for a further 30 days.
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| Growth protocol |
HEK 293T cells were grown in triplicates, in DMEM supplemented with 10% fetal bovine serum and 2 mM Penicillin-Streptomycin at 37°C in 5% CO2. Cells were passaged only when reaching 80-90% confluence, for a total period of one month.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
HEK 293T cells were collected by washing and trypsinizing for three minutes. The same procedure was performed with cells allowed to remain in culture for 30 days without the drug. Genomic DNA samples were extracted using standard protocols.
Two custom adapters were created for HELP-tagging, referred to as AE and AS. As well as an Illumina adapter sequence, adapter AE contains an EcoP15I recognition site and a T7 promoter sequence. Adapter AS contains an Illumina sequencing primer sequence. One micrograms of genomic DNA were digested with HpaII and MspI in separate 50 μl reactions and purified by phenol/chloroform extraction followed by ethanol precipitation. The digested genomic DNA was ligated to adapter AE using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, 1 μg of MspI/HpaII-digested DNA , 0.1 μl of Adapter AE (1 μM), 3 μl of Quick Ligase in a final volume of 50 μl). After AE ligation, the products were purified using AMpure (Agencort), then digested with EcoP15I (NEB). The restriction fragments were end-repaired to inhibit dimerization of adapters, and tailed with a single dA, at the 3’ end. After the dA tailing reaction, adapter AS was ligated to the dA-tailed fragments using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, 2.5 μl of Adapter AS (10 μM), 2.5 μl of Quick Ligase in a final volume 50 μl). After ligation, products were purified using the MinElute PCR purification kit (Qiagen) and in vitro-transcribed using MEGAshortscript (Ambion). Following in vitro transcription, products were purified with an RNeasy clean-up kit (Qiagen) before reverse transcription was performed using the SuperScript III kit (Invitrogen). The first strand cDNA produced was used as a template for PCR using the following conditions: 98°C for 2 minutes, then 18 cycles of 98°C for 15 seconds, 60°C for 15 seconds and 72°C for 15 seconds followed by 5 minutes at 72°C for the final extension. The product then underwent gel extraction (Qiagen).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Library strategy: HELP-tagging
Images generated by the Illumina sequencer were analyzed by Illumina pipeline software (versions ELAND - 1.7.0). Initial data processing was performed using the default read length of 36 bp. We permitted up to two mismatches in each sequence, and allowed a sequence to align to up to a maximum of 10 locations within the genome. For non-unique alignments, a sequence was assigned a partial count for each aligment location amounting to 1/n, where n represents the total number of aligned positions. To normalize the data between experiments, the number of sequences associated with each HpaII site was divided by the total number of sequences (including partial counts) aligning to all HpaII sites in the same sample.
Genome_build: hg19
Supplementary_files_format_and_content: The wig files were generated using in house pipeline; Scores represent degree of unmethylation
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| Submission date |
Oct 22, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Maria-Paz Ramos |
| Organization name |
Albert Einstein College of Medicine
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| Department |
Genetics
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| Lab |
John Greally
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| Street address |
1301 Morris Park Ave, Price 314
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| City |
Bronx |
| State/province |
New York |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE62590 |
DNA demethylation by 5-aza-2'-deoxycytidine is imprinted targeted to euchromatin and has limited transcriptional consequences |
| GSE62592 |
HEK 293T cells HELP-tagging cytosine methylation data treated with 5-aza-2-deoxycytidine |
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| Relations |
| BioSample |
SAMN03135176 |
| SRA |
SRX738254 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1529695_hg19.H_100.AC1C8MACXX.lane_8.AH81WVADXX.lane_2_hg19.M_NT_R.AC1C8MACXX.lane_8.AH81WVADXX.lane_2.wig.gz |
8.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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