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| Status |
Public on Jul 23, 2014 |
| Title |
hESp0-E1 |
| Sample type |
SRA |
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| Source name |
ESC outgrowth
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| Organism |
Homo sapiens |
| Characteristics |
developmental stage: ESC outgrowth
passage: 0
sequenced molecule: Msp1 digested bisulfite converted genomic DNA
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| Treatment protocol |
hICM, hTE,mICM and mTE were isolated using standard micromanipulation equipment and a Hamilton Thorne XYClone laser with 300 µs pulsing at 100% intensity. hBlast and 8c embryos were thawed using the Quinn’s Advantage Embryo Thaw Kit (SAGE), screened for morphology, and purified from the Zona Pellucida using Acid Tyrode's solution (Sigma). Human sperm was purified via centrifugation through a PureCeption gradient solution (PureCeption 100% Isotonic Solution, Quinn’s Advantage Medium with HEPES, In-Vitro Fertilization Inc) of 90% and 47% in a 15ml conical tube. C57Bl/6 x 129S1/SvIMJ E6.5 Epi and ExE were dissected from implanted deciduae and purified of visceral endoderm via incubation in 0.5% trypsin/2.5% pancreatin (Sigma) dissolved in PBS for 20 minutes at 4ºC.
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| Growth protocol |
hESCs were derived and cultured on gamma-irradiated mouse embryonic fibroblasts (MEFs) in derivation media consisting of KO-DMEM (Life Technologies), 15% KO-SR (Life Technologies), 2.5% Fetal Bovine Serum (FBS) (Hyclone), 2mM Glutamax, 1% non-essential amino acids, 50units/ml penicillin and 50μg/ml streptomycin (Life Technologies), 0.055mM β-mercaptoethanol (Life Technologies), 10ng/ml bFGF (Millipore). mESCs were derived from immunosurgically isolated ICM of E3.5 blastocysts from C57Bl/6 x 129S1/SvIMJ hybrid crosses and propogated on gamma-irradiated MEFs in one of three conditions: mES - 15% Fetal Bovin Serum (Hyclone) supplemented KO-DMEM plus LIF (Millipore); mPD - 15% FBS supplemented KO-DMEM plus LIF and 1 µM PD0325901 (Stemgent), or in N2/B27 supplemented KO-DMEM containing LIF, 1 µM PD0325901 and 3 µM CHIR99021 (Stemgent) (m2i). All mouse media were additionally supplemented with 2mM Glutamax, 1% non-essential amino acids, 50units/ml penicillin and 50μg/ml streptomycin (Life Technologies), 0.055mM β-mercaptoethanol (Life Technologies).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from all tissue using 14 hour incubation in 300 ug/mL Proteinase K followed by ethanol precipitation and elusion in EB buffer
Reduced representation bisulfite libraries were prepared from Proteinase K treated, MspI digested DNA following ligation with methylated standard adapters, size selection, bisulfite conversion, and amplification of the final libraries. Reduced representation bisulfite sequencing libraries are designed to enrich for a consistent subset of CpGs in the genome using MspI digestion (C'CGG cutting), adapater ligation and bisulfite conversion to generate base resolution maps of cytosine methylation on a consistent genome scale
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
RRBS; Raw data not supplied for human subjects due to privacy concerns
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| Data processing |
Alignment using Maq with the following params: maq map -D -s 0 -M c -e 100
Count the number methylated vs unmethylated CpGs (C vs T; G vs A on reverse)
Join the cpg files together
Add the CpG density column (#CpGs +/- 50bp)
Tile the cpg file
Average technical replicates
Supplementary_files_format_and_content: .cpgs: (chromosome,position,seenCount,methylatedCount);hDev100bpMeFinal.txt/mDer100bpMe.txt: (position,100bp methylation value,CpG density)
Genome Build: hg19
Raw data not supplied for human subjects due to privacy concerns
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| Submission date |
Sep 27, 2013 |
| Last update date |
Jul 23, 2014 |
| Contact name |
Michelle M Chan |
| Organization name |
UCSF
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| Lab |
Weissman Lab
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| Street address |
1700 4th St.
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE51239 |
DNA methylation dynamics of the human pre-implantation embryo |
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| Relations |
| BioSample |
SAMN02364716 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1240875_hESp0-E1.cpgs.txt.gz |
15.0 Mb |
(ftp)(http) |
TXT |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
| Processed data are available on Series record |
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