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Sample GSM1954805 Query DataSets for GSM1954805
Status Public on Feb 28, 2017
Title Stategra_Ikaros-ERt2 cells 0h 4-OHT Batch4
Sample type SRA
Source name pre-B lymphocyte
Organism Mus musculus
Characteristics transfection: inducible Ikaros
time: 0h
#replicate: 3
#batch: 4
treatment: 4-OHT
dose: 0.5 uM
cell line: B3
cell type: pre-B lymphocyte
Treatment protocol Cells containing inducible Ikaros were generated by transducing mouse pre-B cell line B3 with mouse stem cell virus (MSCV) retroviral vectors encoding a fusion protein of haemagglutinin-tagged wild type Ikaros (HA-Ikaros) and the estrogen receptor hormone-binding domain (ERt2), followed by an internal ribosomal entry site (IRES) and GFP. B3 cells containing inducible Ikaros (HA-Ikaros-ERt2-IRES-GFP) were plated at a density of 0.5 million cells/ml in IMDM medium containing 10% FCS and 1% penicillin-streptomycin. 0.5uM 4 hydroxy-tamoxifen was added to the cells 2h before collection. Control vector-ERt2 B3 cells were plated at a density of 0.5 million cells/ml in IMDM medium supplemented with 10% FCS and 1% penicillin/streptomycin.
Extracted molecule total RNA
Extraction protocol Time point samples were collected by 5 min centrifugation at 1200 rpm. Cell pellets were washed 2 times in PBS. RNA from all samples was extracted usng RNAbee (AMS Biotechnology (Europe) Ltd) and treated with Turbo DNase (Life Technologies). Agilent Bioanalyzer was used to check the RNA integrity and samples were quantified using a Nanodrop
Total RNA with RNA Integrity number >8 (Agilent Bioanalyzer) was quantified using a Nanodrop. Stranded mRNA Libraries were prepared as follows, mRNA was isolated from 4 micrograms of total RNA using oligo dT beads, fragmented and cDNA was synthesised. 2nd strand cDNA was synthesised and labelled with dUTPs . This was followed by adapter ligation with barcoded Illumina adapters and 14 cycles of PCR amplification of adapter ligated DNA. Libraries were quantified using the Qubit Nano DNA quantification kit. Molar concentration of libraries was done with the Kapa Illumina Quantification Kit. 14pM of pooled libraries was sequenced per lane on the HiSeq 2500 with paired end, 75 bp sequencing.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Description 0h after Taxomifen induction in Ikaros/ERt2 cells
Data processing Trimming was applied to remove Illumina primers and low-quality nucleotides.; the very-sensitive mode only allowing a unique best mapping per fragment was used.
Tophat2 (Kim et al 2013) was used to map reads to the mm10 reference genome
Htseq-count (intersect option) was used to assign fragments to genes.
Counts were normalized with cqn (Hansen et al, 2012).
ComBat was used to reduce batch effect
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file containing mRNA quantification values for each Sample (matrix).
Submission date Nov 25, 2015
Last update date May 15, 2019
Contact name Ana Conesa
Organization name Centro de Investigaciones Príncipe Felipe (CIPF)
Department Computational Genomics Program
Lab Genomics and Gene Expression Lab
Street address C/ Eduardo Primo Yúfera 3
City Valencia
State/province Valencia
ZIP/Postal code 46012
Country Spain
Platform ID GPL17021
Series (2)
GSE75395 Omics analyses of B3 pre-B cell line from STATegra Project
GSE75417 mRNA-seq analysis of B3 pre-B cell line from STATegra Project
BioSample SAMN04296331
SRA SRX1452626

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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