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| Status |
Public on Aug 01, 2012 |
| Title |
fetal RPE |
| Sample type |
SRA |
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| Source name |
fetal RPE
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| Organism |
Homo sapiens |
| Characteristics |
cell type: primary fetal RPE
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| Growth protocol |
Pluripotent stem cells (hESC and iPSCs) were plated onto gamma-rays irradiation mouse embryonic feeder cells with DMEM/F12 culture medium containing 20% Knock-Out Serum Replacement, 0.1 mM nonessential amino acids, 0.1 mM b-mercaptoethanol and 100 ng/ml zebrafish basic fibroblast growth factor (zfbFGF) on a 6-well plate. Briefly, cells were cultured at 37°C in 5% CO2 for 6-10 days after which zfbFGF was omitted to facilitate spontaneous cell differentiation.Pigmented colonies were observed within 4 weeks and allowed to expand for a few weeks, with media changes every 2–3 days. Pigmented cells were enriched by manual dissection using insulin needle followed by seeded on growth factor reduced Matrigel (BD Biosciences, diluted 1:30) coated plate and transwell membranes. RPE medium were changed to support pigment cluster expansion[containing a-MEM, 1 x N2 supplement(Gibco), 1x Non-essential amino acid solution, 250 mg/ml taurine, 13 ng/ml Triiodo thyronin (Sigma-Aldrich, Gillingham, UK), 20 ng/ml Hydrocortisone (Sigma), 2mM L298 glutamine (Invitrogen, Paisley,UK), 1x Penicillin-streptomycin and 10% Hyclone heat inactivated foetal bovine serum(Thermo Scientific, Northumberland, UK)], which was replaced daily. After 2 to 3 months，under these conditions, hESC-RPE and hiPSC RPE would form monolayer sheets of pigmented cells that can be dissected for gene expression analysis.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (1~10μg) was ligated with a pair of Illumina adaptors to their 5′and 3′ends. After reverse transcription, the small RNA molecules were amplified using multiplex adaptor primers for 12 cycles and the fragments around 93~100 bp (small RNA+adaptors) were then isolated from agarose gel. The purified DNA was used directly for cluster generation and sequencing analysis using the Illumina's HiSeq2000 sequencing according to the manufacturer's instructions.
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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 |
Sequenced reads were trimmed for adaptor sequence, then mapped to hg19 whole genome using bowtie v0.12.2 with parameters -v1 -a -m 10 --best --strata
Reads Per Megabase of library size (RPM) were calculated by counting the number of reads falling in the miRNA locus and normalizing by the size of the library.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPM values for each sample
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| Submission date |
May 01, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kevin Huang |
| Phone |
310-267-0438
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| Organization name |
UCLA
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| Department |
Human Genetics
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| Lab |
Guoping Fan
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| Street address |
Gonda BLDG Rm. 6554, P.O.Box 957088
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095-7088 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE37686 |
Identification of miRNA signatures during the differentiation of hESCs into retinal pigment epithelial cells |
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| Relations |
| SRA |
SRX145386 |
| BioSample |
SAMN00990523 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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