|
| Status |
Public on May 18, 2015 |
| Title |
Day 15 +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 1.17.21.3) 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 |
| E-mail(s) |
munazah@cdb.riken.jp
|
| 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 |
|
| Relations |
| BioSample |
SAMN03113001 |
| SRA |
SRX734337 |
