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Sample GSM5224984 Query DataSets for GSM5224984
Status Public on Mar 30, 2022
Title EL4 T-bet cells unstimulated
Sample type SRA
 
Source name EL4 cell line
Organism Mus musculus
Characteristics cell line: EL4
treatment: unstimulated
ectopically expressed protein: FLAG-T-bet
Treatment protocol EL4 cells were stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin at 37°C for 4 hrs, or left unstimulated
Growth protocol EL4 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 4 mM L-alanyl-L-glutamine, 25 mM D-glucose, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin and 100 µg/ml streptomycin
Extracted molecule genomic DNA
Extraction protocol 50,000 cells were washed in 50 μl cold PBS (500g, 5 min, 4°C) and re-suspended in 50 μl cold lysis buffer I (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% IGEPAL CA-630) and spun (500g, 10 min, 4°C). The supernatant was removed, and lysed cells re-suspended in 50 μl transposase reaction mix, containing 10 mM Tris pH8.0, 5 mM MgCl2, 10% dimethylformamide and 2.5 μl Tn5 transposon + transposase reagent (TDE1 Illumina). Reactions were incubated at 37°C for 45 min, with shaking at 500 rpm. Following transposition, 150 μl reverse-crosslinking buffer (50 mM Tris-Cl, 1 mM EDTA, 1% SDS, 0.2 M NaCl) was added to reactions, and incubated at 65°C overnight at 1000 rpm before DNA purification, using the MinElute PCR purification kit (Qiagen).
Transposed DNA was amplified by PCR for 5 cycles, using KAPA HiFi HotStart ReadyMix, 1.25 μM Ad1 primer and 1.25 μM Ad2.x indexed primers, as described (Buenrostro et al., 2013). 5 μl of each reaction was taken for qPCR as described, using KAPA HiFi HotStart ReadyMix and 50X KAPA Low ROX for signal normalisation, to determine the additional number of PCR cycles for library amplification. PCR reactions were purified using MinElute gel extraction kit (Qiagen) according to the manufacturer’s instruction, and fragments between 100-1000 bp in size were selected for sequencing. DNA Fragmentation of the library was measured using the Bioanalyzer High Sensitivity DNA assay (Agilent), and library concentration quantified by qPCR analysis, using Illumina Library Quantification kit (KAPA). DNA from multiple libraries was pooled at equimolar concentrations to a final concentration of 4 nM.
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina HiSeq 3000
 
Data processing Pooled libraries were sequenced on HiSeq 3000 (Illumina) using 75 bp paired-end reads
Raw reads were filtered for base quality using FastQC with default parameters and adapters sequences were removed with Trim Galore!
Reads passing quality filtering criteria were aligned to GRCm38 using BWA-MEM with default settings
Genome_build: mm10
Supplementary_files_format_and_content: bigWig
 
Submission date Apr 02, 2021
Last update date Mar 30, 2022
Contact name Arnulf Hertweck
E-mail(s) a.hertweck@ucl.ac.uk
Organization name University College London
Department UCL Cancer Institute
Street address Gower Street
City London
ZIP/Postal code WC1E 6BT
Country United Kingdom
 
Platform ID GPL21493
Series (2)
GSE171407 The TH1 cell lineage-determining transcription factor T-bet supresses TH2 gene expression by redistributing GATA3 away from TH2 genes [ATAC-seq]
GSE171410 The TH1 cell lineage-determining transcription factor T-bet supresses TH2 gene expression by redistributing GATA3 away from TH2 genes
Relations
BioSample SAMN18605321
SRA SRX10505860

Supplementary file Size Download File type/resource
GSM5224984_EL4-Tbx21_US.bigWig 204.7 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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