|Public on Feb 28, 2017
|Stategra_Ikaros-ERt2 cells 0h 4-OHT Batch4
|transfection: inducible Ikaros
dose: 0.5 uM
cell line: B3
cell type: pre-B lymphocyte
|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.
|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.
|Illumina HiSeq 2500
|0h after Taxomifen induction in Ikaros/ERt2 cells
|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
Supplementary_files_format_and_content: tab-delimited text file containing mRNA quantification values for each Sample (matrix).
|Nov 25, 2015
|Last update date
|May 15, 2019
|Centro de Investigaciones Príncipe Felipe (CIPF)
|Computational Genomics Program
|Genomics and Gene Expression Lab
|C/ Eduardo Primo Yúfera 3
|Omics analyses of B3 pre-B cell line from STATegra Project
|mRNA-seq analysis of B3 pre-B cell line from STATegra Project