|
| Status |
Public on Dec 06, 2011 |
| Title |
Ribominus total RNA from ES 6 |
| Sample type |
SRA |
| |
|
| Source name |
Day5 differentiated CCE
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129/SvEv
cell type: differentiated ES cells induced by retinoic acid for 5 days
fragmentation method: RNA hydrolysis
rrna depletion: 1 round
sequencing facility: NCMLS Solexa Sequencing facility, Nijmegen
|
| Growth protocol |
CCE feeder-independent ES cells were grown under standard conditions, and differentiation was induced by withdrawal of LIF and addition of 0.266µM retinoic acid (RA). Mouse fetal heads were dissected at 14.5 days postcoitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DNase I treated 10µg of high quality total RNA from cells or tissues was subjected to ribosomal RNA depletion by using the RiboMinus Transcriptome Isolation Kit (Human/Mouse) according to the manufacturer’s protocol. The quality of the purified RNA was checked on Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit according to the manufacturer’s protocol. The purified Ribominus RNA fraction (100ng) was subsequently fragmented by addition of 5× fragmentation buffer (200mM Tris pH8.2, 500mM KAc and 150mM MgAc2) and heating at 94°C for 3.5min in a total volume of 100µl. The reaction was stopped by putting the sample on ice. The fragmented RNA was purified by RNeasy Mini Kit according to the manufacturer protocol, and subsequently ethanol precipitated and dissolved in 10µl nuclease-free ddH2O.The fragmented RNA was used as template for cDNA synthesis using 5µg random hexamers in a total volume of 20µl according to the Superscript II Reverse Transcriptase standard protocol. Second-strand synthesis was performed by adding 91.8µl water, 30µl 5× Second strand buffer, 3µl 10mM dNTP, 4µl DNA polymerase I, 1µl E. coli DNA ligase and 0.2µl RNase H, followed by incubation at 16°C for 2hour. T4 DNA Polymerase (1µl) was added followed by an additional 10 min at 16 °C. The ds-cDNA was purified by using the MinElute Reaction Cleanup Kit according to the manufacturer’s protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Ribominus total RNA, Trizol isolated
|
| Data processing |
Alignment: Sequence reads were obtained using the Illumina Genome Analyzer Pipeline and mapped to the mouse genome (mm9, July 2007) using Bowtie. All reads mapping with two or fewer mismatches were retained.
|
| |
|
| Submission date |
Jul 15, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Florian M Pauler |
| E-mail(s) |
florian.pauler@ist.ac.at
|
| Phone |
+43 2243 9000-7434
|
| Organization name |
IST Austria
|
| Lab |
Simon Hippenmeyer
|
| Street address |
Am Campus 1
|
| City |
Klosterneuburg |
| ZIP/Postal code |
3400 |
| Country |
Austria |
| |
|
| Platform ID |
GPL9185 |
| Series (1) |
| GSE22959 |
Sequencing of the non-ribosomal transcriptome allows the simultaneous identification of protein-coding and non-protein-coding RNAs |
|
| Relations |
| SRA |
SRX023797 |
| BioSample |
SAMN00017240 |
