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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 20, 2012 |
| Title |
H9 |
| Sample type |
SRA |
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| Source name |
embryonic stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: embryonic stem cells
passages: 29
cell line: H9
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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 |
total RNA |
| Extraction protocol |
4μg of the total RNA isolated from each sample was used for digital gene expression (DGE) library construction. Briefly, the total RNA was treated with Oligo(dT) magnetic bead to purify mRNA and then synthesized double-stranded cDNA using Oligo(dT) as primer. The cDNA was digested with NlaIII and ligated to a first adapter containing a restriction site recognized by MmeI. After dephosphorylation with alkaline phosphatase CIAP, the purified MmeI-digested products were linked to a second adapter. Then, the double adapter-flanked tags from the mRNAs were amplified by PCR using Phusion DNA polymerase and Gex PCR primers following the manufacturer’s protocol. PCR was carried out using the following program: 98℃ for 30s, followed by 15 cycles of 98℃ for 10 s, 60℃ for 30 s and 72℃ for 15 s, and then 72℃ for 10 min. The resulting～95-bp PCR products were purified by 6% TBE PAGE electrophoresis gels using Spin-X filter columns. Finally, mRNA libraries were sequenced on the Illumina HiSeq 2000 following the manufacturer’s protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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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. Clean reads were aligned to the hg19 genome assembly using SOAPaligner version 2.21 with no more than one mismatch
Tags of gene per Megabase of library size (TPM) were calculated using a protocol from Hoen, Ariyurek, et al., Nucleic Acids Research, 2008; Morrissy, Morin, et al. Genome Research, 2009.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each sample.
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| Submission date |
Sep 18, 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) |
| GSE40953 |
Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation [DGE] |
| 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 |
SRX189167 |
| BioSample |
SAMN01728916 |
| 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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