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Sample GSM1954358 Query DataSets for GSM1954358
Status Public on Feb 28, 2017
Title STATegra Control cells 12h Batch1
Sample type SRA
 
Source name pre-B lymphocyte
Organism Mus musculus
Characteristics transfection: control
time: 12h
#replicate: 1
#batch: 1
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.
Extracted molecule genomic DNA
Extraction protocol 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.
 
Library strategy Bisulfite-Seq
Library source genomic
Library selection Reduced Representation
Instrument model Illumina HiSeq 2500
 
Description 12h after Taxomifen induction in Control cells
Data processing 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
Genome_build: mm10
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).
 
Submission date Nov 25, 2015
Last update date May 15, 2019
Contact name Ana Conesa
E-mail(s) info@stategra.eu
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)
GSE75393 Methyl-seq analysis of B3 pre-B cell line from STATegra Project
GSE75395 Omics analyses of B3 pre-B cell line from STATegra Project
Relations
BioSample SAMN04295772
SRA SRX1452122

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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