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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 17, 2012 |
| Title |
E14Tg2a |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain: 129/Ola
cell line: E14Tg2A
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| Treatment protocol |
Genomic DNA library coupled with protection of 5-hmC, Tet-assisted 5mC oxidation, and bisulfite treatment
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| Growth protocol |
E14 (E14Tg2A) ES cell lines were cultured in feeder-free gelatin-coated plates in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen Cat. No. 11995) supplemented with 15 % FBS (GIBCO), 2 mM L-glutamine (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma), 1x non-essential amino acids (GIBCO), 1,000 units/ml LIF (Millipore Cat. No. ESG1107), 1x pen/strep (GIBCO). The culture was passaged every 2 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Purified genomic DNA was fragmented to an average size of 300bp with a Covaris S2 (10% duty cycle, intensity 4, 200 cycles per burst, 80 sec). Spike in 5mC conversion controls were added prior to sonication at 0.5% of total genomic DNA. Glycosylation reactions were performed in a 50 μl solution containing 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, 100 ng/μl sonicated humun or mouse genomic DNA, 200 μM UDP-Glc, and 1 μM wild-type βGT. The reactions were incubated at 37 oC for 1 h. After the reaction, the DNA was purified by QIAquick Nucleotide Removal Kit (Qiagen). The oxidation reactions were performed in a 50 μl solution containing 50 mM HEPES buffer (pH 8.0), 100 μM ammonium iron (II) sulfate, 1 mM α-ketoglutarate, 2 mM ascorbic acid, 2.5 mM DTT, 100 mM NaCl, 1.2 mM ATP, 10 ng/μl glycosylated DNA and 3 μM recombinant mTet1. The reactions were incubated at 37 oC for 1.5 h. After proteinase K treatment, the DNA was purified with Micro Bio-Spin 30 Columns (Bio-Rad) first and then applied to QIAquick PCR Purification Kit (Qiagen). 500ng-1ug TABS treated DNA was end-repaired, adenylated, and ligated to methylated (5mC) adapters (Illumina TruSeq Genomic DNA adapters) according to standard Illumina protocols for genomic DNA library construction, maintaining the proper molar ratios of adapter to insert. Adapter ligated fragments with 200-400bp inserts were gel purified by 2% agarose gel electrophoresis and sodium-bisulfite treated using the MethylCode kit (Invitrogen). Bisulfite treated adapter-ligated DNA was amplified by PCR with the following conditions in a 50uL final reaction volume: 2.5U PfuTurbo Cx Hotstart DNA polymerase, 5uL 10X PfuTurbo Cx reaction buffer, 1.25uL 10mM dNTPs, 5uL Illumina TruSeq PCR Primer Cocktail. Cycling parameters: 95ºC 5min, 98ºC 30 sec, followed by 7-9 cycles of 98ºC 10 sec, 65ºC 30 sec, 72ºC 30 sec, and ending with 72ºC 5 min. The number of PCR cycles used was determined by quantification of bisulfite treated adapter-ligated DNA by qPCR (KAPABiosystems library quant kit for Illumina libraries) such that the final library concentration obtained was approximately 20nM. Final sequencing libraries were purified with AMPure XP beads and quantified by qPCR (KAPABiosystems library quant kit for Illumina libraries). Up to 3 separate PCR reactions were performed per sample.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
5-hmC whole genome bisulfite sequencing
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| Data processing |
Reads were processed as previously reported(Hon et al., 2012; Lister et al., 2009). Briefly, raw reads were trimmed for low quality bases and adapter sequences. Then, cytosine bases were computationally replaced with thymines, mapped with the Bowtie program (Langmead et al., 2009) against computationally converted copies of hg18 or mm9, and mapped reads were resorted to their pre-computationally-converted bases. PCR duplicates were removed for each PCR amplification reaction using the Picard program (http://picard.sourceforge.net). To eliminate reads not bisulfite converted, reads having more than 3 base calls in non-CG context were removed, as previously (Lister et al., 2009). All libraries were then merged and indexed by the SAMtools suite (Li et al., 2009).
Genome Build:
mESC.mm9.+.bed.gz: mm9
mESC.mm9.-.bed.gz: mm9
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| Submission date |
Feb 29, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Gary Chung Hon |
| Organization name |
UT Southwestern
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| Department |
OB/GYN
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE36173 |
Base Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome |
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| Relations |
| SRA |
SRX131671 |
| BioSample |
SAMN00839608 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM882244_calls_readme.txt.gz |
1020 b |
(ftp)(http) |
TXT |
| GSM882244_mESC.hmC_sites.FDR_0.0484.mm9.txt.gz |
14.6 Mb |
(ftp)(http) |
TXT |
| GSM882244_mESC.mm9.+.bed.gz |
134.9 Mb |
(ftp)(http) |
BED |
| GSM882244_mESC.mm9.-.bed.gz |
133.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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