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| Status |
Public on Jun 04, 2014 |
| Title |
mTET1-CD_DREAM |
| Sample type |
SRA |
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| Source name |
HEK293T cells
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| Organism |
Homo sapiens |
| Characteristics |
transfection: mTET1-CD
cell sorting time: 3 days after transfection
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| Growth protocol |
HEK293T cells were cultured in Dulbecco's Modified Eagle Medium modified with 10% fetal bovine serum and 100 μg/ml streptomycin-penicillin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
mTET1-CD, TET1-CD, mTET1-FL or TET1-FL expression plasmid (containing GFP reporter) was transfected into HEK293T cells by using OPTI-MEM (Invitrogen) and FuGene HD transfection reagent (Roche), followed by fluorescent activated cell sortng to collect GFP positive cells 3 (for mTET1-CD or TET1-CD ) or 7 (for mTET1-FL or TET1-FL ) days after transfection. The genomic DNA was then extracted from those cells.
Five micrograms of genomic DNA spiked with 5 methylation standards with defined methylation levels of 0, 25, 50, 75, and 100% were digested with 100 units of SmaI endonuclease (NEB) for 3 hours at 25°C. Subsequently, 100 units of XmaI endonuclease (NEB) were added and the digestion was continued for additional 16 hours at 37°C. Digested DNA was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted in TRIS-HCl 10 mM pH 8.5 (EB). Eluted DNA was supplemented with NEB buffer #2, dCTP, dGTP and dATP (0.4 mM final concentration of each), 15 units of Klenow Fragment (3´→5´ exonuclease deficient) DNA polymerase (NEB) and incubated for 30 minutes at 37°C. This step filled in the recesses at 3’ DNA ends created by XmaI digestion and added 3’ dA tails to all fragments. Solexa paired ends adapters were then ligated at 10:1 adapter:fragment ratio using Rapid T4 DNA ligase (Enzymatics, Beverly, MA). The ligation mix was size selected by electrophoresis in 2% agarose. Two slices corresponding to 250-375 bp sizes based on a 100 bp DNA ladder (NEB) were cut out and DNA was extracted from agarose. DNA eluted from the slices was separately amplified with Solexa paired end PCR primers using iProof high-fidelity DNA polymerase (Bio-Rad Laboratories, Hercules, CA) and 18 cycles of amplification. Resulting sequencing libraries were purified with AMPure magnetic beads (Agencourt, Beverly, MA) and then ready for paired-end sequencing on Illumina Genome Analyzer II.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Sequencing tags were mapped to SmaI sites in the human genome (hg18) and signatures corresponding to methylated (starting with CCGGG…) and unmethylated CpG (starting with GGG…) were enumerated for each SmaI/XmaI site.
The methylation value was calculated as the ratio of the number of tags starting with CCGGG over total number of tags mapped to a given SmaI/XmaI site.
Genome_build: hg18
Supplementary_files_format_and_content: The columns of the processed file are: SmaI_ID(SmaI site ID), Chrom(chromosome of the SmaI site), Pos(position of the SmaI site), MetReads(observed number of tags corresponding to methylated CpG), UnmetReads(observed number of tags corresponding to unmethylated CpG), Methylation(methylation value).
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| Submission date |
Feb 04, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
CHUNLEI JIN |
| E-mail(s) |
jincl1980@gmail.com
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| Organization name |
The University of Texas MD Anderson Cancer Center
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| Street address |
1515 Holcombe Blvd
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (2) |
| GSE44038 |
TET1 is a maintenance DNA demethylase that prevents methylation spreading in adult cells [DREAM] |
| GSE44039 |
TET1 is a maintenance DNA demethylase that prevents methylation spreading in adult cells |
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| Relations |
| SRA |
SRX224088 |
| BioSample |
SAMN01919543 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1077162_mTET1-CD_DREAM.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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