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Sample GSM925605 Query DataSets for GSM925605
Status Public on Aug 01, 2012
Title ESC BG01 rep1
Sample type SRA
 
Source name ESC
Organism Homo sapiens
Characteristics cell type: ESC
cell line: BG01
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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
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
 
Submission date May 01, 2012
Last update date May 15, 2019
Contact name Kevin Huang
Phone 310-267-0438
Organization name UCLA
Department Human Genetics
Lab Guoping Fan
Street address Gonda BLDG Rm. 6554, P.O.Box 957088
City Los Angeles
State/province CA
ZIP/Postal code 90095-7088
Country USA
 
Platform ID GPL11154
Series (1)
GSE37686 Identification of miRNA signatures during the differentiation of hESCs into retinal pigment epithelial cells
Relations
SRA SRX145383
BioSample SAMN00990520

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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