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| Status |
Public on Jun 04, 2014 |
| Title |
mTET1-CD-Input |
| 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
fraction: Input
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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.
Before hydroxymethylated DNA immunoprecipitation (hMeDIP) reaction, genomic DNA samples were sonicated into a desirable fragment size of 100~500 bp. The purified sonicated DNA was then prepared for adapter ligation as per Illumina’s standard protocol (Illumina, San Diego, CA) with minor modifications. In brief, DNA was end-repaired using a combination of T4 DNA polymerase, Klenow enzyme (DNA Polymerase I Large Fragment) and T4 polynucleotide kinase (NEB, Ipswich, MA). The blunt phosphorylated DNA fragments were treated with Klenow fragment (3’ to 5’ exo minus) (NEB) and dATP to add an ‘A’ base to the 3’ end. Solexa paired ends adapters were ligated to the ends of the DNA fragments using Quick T4 DNA Ligase (Enzymatics, Beverly, MA). The resulting DNA products were subsequently subjected to hMeDIP for purifying 5hmC-containing DNA fragments. The immunoprecipitated and input control DNA were size selected by electrophoresis in a 2% agarose gel. A slice corresponding to 300 (± 25) bp size window based on DNA ladder was cut out. The DNA extracted from agarose was amplified with PCR (10-15 cycles) by using Solexa paired end PCR primers and Phusion™ High-Fidelity DNA Polymerase (NEB). The resulting sequencing library was finally cleaned with AMPure magnetic beads (Agencourt, Beverly, MA) and ready for single-read sequencing on Illumina Genome Analyzer II.
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| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Sequenced DNA tags were mapped to human genome hg18 using ELAND (Illumina Analysis Pipeline) and uniquely mapped tags were kept. To avoid PCR bias, for the multiple tags that were mapped to the same genomic location, only one copy was kept. CCAT (version 3.0) was used to detect peaks in hMeDIP-Seq samples. The window size was set as 500 bp. Peaks with FDR ≤ 0.05 and fold enrichment to input ≥ 5 were called significant.
Genome_build: hg18
Supplementary_files_format_and_content: The columns of the processed file are: chr(chromosome of the peak), start(start postion of the peak), end(end position of the peak), tags_chip(number of tags in hMeDIP sample), tags_control(number of tags in Input sample), fold_change(fold change of hMeDIP to Input), FDR(false discovery rate).
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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) |
| GSE44036 |
TET1 is a maintenance DNA demethylase that prevents methylation spreading in adult cells [MeDIP-Seq] |
| GSE44039 |
TET1 is a maintenance DNA demethylase that prevents methylation spreading in adult cells |
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| Relations |
| SRA |
SRX224092 |
| BioSample |
SAMN01909354 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data not applicable for this record |
| Raw data are available in SRA |
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