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| Status |
Public on Dec 20, 2012 |
| Title |
EB_rep1_BGT |
| Sample type |
SRA |
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| Source name |
embryoid body cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: ES-derived embryoid body cells
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| Growth protocol |
For maintenance of H9 hESCs, MEF feeder layer was obtained from CF-1 mouse embryos and cultured in MEF medium, which is composed of 90% DMEM (GIBCO 12430) supplemented with 10% FBS (Biochrom S0615). Before freezing cells, MEFs were cultured for 3h at 37℃ in mitomycin C (Sigma M0503) medium that is composed of MEF medium supplemented with 10 ug/ml mitomycin C. For culturing H9 ES cells, MEF-conditioned medium was produced by conditioning MEFs for at least 24 hours in the medium composed of DMEM/F12 (GIBCO 11330) supplemented with 20% knockout serum replacement (GIBCO 10828), 2 mM L-glutamine (GIBCO 25030), 2 mM nonessential amino acids (GIBCO 11140), 0.1 mM 2-mercaptoethanol (GIBCO 21985-023), and 4 ng/ml recombinant human fibroblast growth factor-basic (bFGF; GIBCO 13256-029). Prior to differentiation, hESCs were cultured on MEFs that were prepared 24 hours in advance, transferred from MEFs onto Matrigel (Invitrogen,354234), and cultured in MEF-conditioned medium. Clumps of hESCs (~5 million cells per plate) were then plated in suspension onto ultra-low adhesion dishes (Corning) in DMEM (Gibco 12430) containing 20% FBS (Biochrom S0615), which promoted the formation of embryoid bodies (EBs).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from H9 hESCs and EBs using QIAamp DNA Blood Mini Kit (QIAGEN)
HSMT-Seq library construction followed the same protocol from Li, J. et al. BMC Genomics, 2009., except that three independent libraries with initially different enzyme digestion were constructed for each sample, which were termed “C” library, “C+mC” library and “C+mC+hmC”, respectively. For “C+mC” library construction, an aliquot of genomic DNA from each sample was first glucosylated by incubating 1 µg of DNA substrates with 3 μ l (30 units) of T4 β-glucosyltransferase (β-GT) (NEB) for 16 hour at 37°C in a total 100 μ l reaction containing 1X NEBuffer 4 supplemented with 80µM UDP-Glc. After glucosylation, the aliquot of glucosylated DNA was then digested with 300 units of MspI (NEB). For the “C” or “C+mC+hmC” libraries, the aliquots of DNA without glucosylation were directly digested with 300 units of HpaII or MspI (NEB), respectively. The enzyme digestion reactions were carried out at 37°C for 16 hours in a 50 μl of either NEBuffer 1 (HpaII) or NEBuffer 4 (MspI). After digestion, the digested aliquots of DNA from all three libraries were ligated with biotinylated linker, fragmented by NlaIII, captured by streptavidin-conjugated beads, digested with MmeI to generate short sequence tags (16-17bp), and ligated with sequencing linkers and amplified by PCR. Then the purified tags were sequenced using Illumina HiSeq 2000 according to the manufacturer’s instructions.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
EB.sites.modification.txt.gz
BGT library (indicating unmodified or methylated cytosines) for replicate 1 of embryoid body cells derived from H9 hESCs
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| Data processing |
Illumina BclConverter-1.9.0 software used for basecalling.
Adaptor sequences were removed, and low-quality sequence reads were trimmed. The clean HMST-seq reads were mapped to hg19 virtual library with no more than one mismatch using open-source programming language Perl.
Average tags of each site for HMST-seq library between replicates were used for further analysis, then a previously published method Global Rank-invariant Set Normalization (GRSN) from Pelz, C.R et al., BMC Bioinformatics, 2008; was adapted for our data normalization among libraries. A rank-invariant set of sites were selected in iterative manner as unmodified “C” tags and used to generate a robust average reference, and lowess algorithm was used to normalize tag counts among all libraries. The hydroxymethylation abundance of specific CCGG site can be determined as the ratio between tag counts of “C+mC+hmC” and “C+mC” libraries. In a similar way, ratio between tag counts of “C+mC” and “C” libraries can represent the methylation abundance. Furthermore, a statistics test based on Poisson distribution was performed between two libraries based on the normalized tag counts to identify significantly modified sites. The CCGG sites possessing significantly different tag counts between two libraries (sequencing depth > 10X, P-value<0.05), meanwhile ratio of tag counts larger than 1, were determined as significantly modified sites with 5-hmC or 5-mC.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include tags abundance, p-value of Poisson test and FDR for each sample.
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| Submission date |
Sep 21, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Desheng Gong |
| E-mail(s) |
gds19870718@163.com
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| Organization name |
Agricultural Genomes Institute at Shenzhen
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| Street address |
No.7 PengFei road
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| City |
Shenzhen |
| ZIP/Postal code |
518120 |
| Country |
China |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE41069 |
Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation [HMST-Seq] |
| GSE41071 |
Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation |
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| Relations |
| SRA |
SRX189177 |
| BioSample |
SAMN01728926 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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