|Public on Feb 28, 2017
|STATegra Ikaros cells 24h 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.
|B3 cells containing inducible Ikaros 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, frozen in liquid nitrogen and storaged at -80. 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. Samples were collected by 5 min centrifugation at 1200 rpm. Cell pellets were washed 2 times in PBS, frozen in liquid nitrogen and storaged at -80.
gDNA concentration was measured with Quant-it Picogreen dsDNA assay and 1 μg input was used in the Msp I digestion. Following overnight incubation at 37°C, digestion reactions were terminated by adding 0.5 M EDTA and purified on a GeneJET PCR purification column. Libraries were prepped using the NEBNext Ultra DNA library preparation kit for Illumina and methylated adapters (Index primers Set 1). Subsequently, adapter ligated fragments were BS converted using the EZ DNA Methylation Gold kit (Zymo Research). 14 cycles of PCR were performed and the products were purified using AMPure XP beads. Quality of the final libraries was checked on a High sensitivity DNA chip (Agilent) and concentration was measured with qPCR. Sequencing was done on an Illumina HiSeq2500 PE 2x100bp.
|Illumina HiSeq 2500
|24h after Taxomifen induction in Ikaros/ERt2 cells
|The FASTQ sequence reads were generated using the Illumina Casava pipeline.
FASTQC quality control tool version 0.10.0
The paired end 100bp sequences reads were mapped using the Bismark package v0.10.1
Quality control for alignments using HTseq QA tool
Calling using the Bismark package v0.10.1
Calling using the MethylKit R package
BedGraph and *.cov files were further considered and analyzed with the BiSeq package
Coverage was inspected before proceeding to smooth the methylation levels (between 0 and 1) per CpG site to avoid potential bias during the smoothing step
Methylation levels were smoothed as described in Hebestreit, K. et al. Bioinformatics 29, 1647–1653 (2013).
M-values were obtained as M = log2(B/(1-B)), where B is the constrained methylation level
Supplementary_files_format_and_content: tab-delimited text files containing Methylation levels for each Sample (matrix) formatted as chr_start_end (i.e. Chromosome number, Start position and End position).
|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
|Methyl-seq analysis of B3 pre-B cell line from STATegra Project
|Omics analyses of B3 pre-B cell line from STATegra Project