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Sample GSM566805 Query DataSets for GSM566805
Status Public on Dec 06, 2011
Title Ribominus total RNA from fetal head 6
Sample type SRA
 
Source name 14.5dpc fetal head
Organism Mus musculus
Characteristics strain: FVB/N
tissue type: fetal head dissected from 14.5dpc mouse embryo
fragmentation method: RNA hydrolysis
rrna depletion: 2 rounds
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 SRX023802
BioSample SAMN00017245

Supplementary file Size Download File type/resource
GSM566805_N_8175_fh2h.BED.gz 11.6 Mb (ftp)(http) BED
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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