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Sample GSM2898780 Query DataSets for GSM2898780
Status Public on Apr 27, 2018
Title Mouse wild type erythroid cells replicate 1
Sample type SRA
Source name Erythroid cells
Organism Mus musculus
Characteristics strain/background: Mixed
genotype/variation: Wild type
cell type: Erythroid
tissue origination: Fetal liver
age: E12.5
Growth protocol Fetal livers were dissected from E12.5 mouse embryos, and the dissociated cell suspension was expanded for 5-7 d in Stempro (Invitrogen) supplemented with Epo (1 U/ml), SCF (50 ng/ml) and dexamethasone (1 uM). After expansion of the cultures, mature red blood cells were removed by negative selection for Ter119 using anti-Ter119 magnetic beads (Miltenyi Biotec), to obtain a population of erythroid precursors. Cultures were then switched to medium containing a high Epo concentration (5 U/ml) to induce differentiation into mature erythroid cells.
Extracted molecule total RNA
Extraction protocol Samples for nascent RNA were collected after 12 h of differentiation. Cells were lysed in TRI Reagent (Sigma), and RNA was extracted according to the manufacturer's instructions. Samples were treated with DNase I using Turbo DNA-free DNase (Ambion). To obtain nascent RNA, fetal liver cultures were pulsed with 50 uM 4-thiouridine (Sigma) for 1 h followed by immediate lysis in TRI Reagent. Nascent RNA was then extracted according to the method described by Dölken et al. (Dölken, L. et al. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA 14, 1959-1972 (2008)).
NEBNext Ultra Directional RNA Library Prep Kit for Illumina.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description Genes analysed: genome-wide
Nascent RNA
Data processing Mapping of reads in tophat (vs1.1.4b), with parameters -r 200 (inner-mate-distance).
Filtering of ribosomal regions (from RepeatMasker), and Blacklisted regions.
Counting genome-wide coverages of read pairs, in 4kb bins.
Transforming the coverages bedgraph to bigwig file (ucsctools bedGraphToBigWig).
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: bigwig file of genome-wide read pair coverage, in 4kb bins.
Submission date Dec 22, 2017
Last update date Apr 27, 2018
Contact name Jelena M Telenius
Organization name Oxford University
Department Weatherall Institute of Molecular Medicine (WIMM)
Lab Genome Biology Research Group
Street address MRC Weatherall Institute of Molecular Medicine University of Oxford John Radcliffe Hospital Headington
City Oxford
ZIP/Postal code OX3 9DS
Country United Kingdom
Platform ID GPL13112
Series (1)
GSE108457 Genetic dissection of the α-globin super-enhancer in vivo [RNA-seq]
BioSample SAMN08226877
SRA SRX3505424

Supplementary file Size Download File type/resource 3.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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