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Sample GSM1526927 Query DataSets for GSM1526927
Status Public on May 18, 2015
Title Day 12 +Wnt, replicate3
Sample type SRA
Source name SFEBq derived Retinal Pigmented Epithelium
Organism Mus musculus
Characteristics strain: EB5 Rx::GFP+/-
tissue: SFEBq derived Retinal Pigmented Epithelium
Treatment protocol At Day 10, GFP+ neural retina epithelium was explanted using forceps and either collected for RNA-seq analysis (NRE samples) or further cultured in retinal maturation media (RMM) containing Wnt/β-catenin signaling stimulating conditions (achieved via inhibition of GSK3 using 3uM CHIR99201 for the first 24 hours and then 1uM CHIR99201 thereafter) or Fgf-signaling stimulating conditions (5ng/mL human recombinant bFgf + 10% FBS) thus generating RPE and NR tissues, respectively. Media was exchanged with fresh media at days 11, 12, and 14. RPE and NR samples were collected in triplicate at Day 12 and Day 15.
Growth protocol ES cell culture was performed using 2i conditioned media (3uM CHIR99201 and 1uM PD184352) in the presence of LIF and blasticidin. ES cells (Rx::GFP cells) were trypsinized and 3000 cells were reaggregated in 100uL differentiation media for each well of a 96-Well low-cell-adhesion plate with Lipidure Coat and incubated at 37C and 5% CO2. Defining trypsinization and reaggregation as Day 1, at Day 2 Matrigel was introduced to achieve a final Matrigel concentration of 4%. Aggregates were incubated at 37C and 5% CO2 until Day 10, when sample collection and Wnt/Fgf treatment began.
Extracted molecule total RNA
Extraction protocol RNA was extracted using the RNeasy kit (Qiagen) using the company-provided protocol. SFEBq aggregates were resuspended in 700uL buffer RLT and spun through QIAshredder (Qiagen) prior to RNA extraction.
Libraries were prepared using 700ng of total RNA, according to the protocol of TruSeq Stranded mRNA LT Sample Prep Kit
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 1500
Data processing Basecalls were performed using illumina RTA software (version and reads were de-multiplexed using bcl2fastq ( version 1.8.4).
Quality check of the reads was performes using FastQC(version 0.10.1). Ascertaining the high quality of sequencing, reads were mappedto mm10 mouse genome assembly using Tophat (version 2.0.8b) with default parameter settings.
Number of reads originating from genes were quantified by cuffdiff program in Cufflinks package (version 2.1.1). These count values were subsequently used to test for differential expression using edgeR(version 3.2.4)
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files including normalized expression values for each sample
Submission date Oct 16, 2014
Last update date May 15, 2019
Contact name Munazah Andrabi
Organization name RIKEN:Center for Developmental Biology
Lab Genome Resource and Analysis Unit
Street address 2-2-3 Minatojima-minamimachi
City Chuo-ku
State/province Kobe
ZIP/Postal code 650-0047
Country Japan
Platform ID GPL18480
Series (1)
GSE62432 Comparative transcriptomic analysis of self-organized, in vitro generated optic tissues
BioSample SAMN03113005
SRA SRX734331

Supplementary file Size Download File type/resource
GSM1526927_plus_Wnt_D12_CPM_rep3.txt.gz 350.1 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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