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Sample GSM998272 Query DataSets for GSM998272
Status Public on Dec 03, 2013
Title Th1_Tbet
Sample type SRA
Source name Th1 cells
Organism Mus musculus
Characteristics antibody: T-bet (Lab produced (Szabo et al., Nature 2000))
cell type: Th1
cell number: 100000000
genotype: wild type
Growth protocol CD4+ T cells from spleens and lymph nodes of 4-10 week-old mice were purified by CD4 positive selection (Miltenyi Biotec) followed by sorting of naïve CD4+CD25-CD62LhighCD44low cells using FACSAria II (BD Biosciences). Cells were activated by plate-bound anti-CD3 and anti-CD28 (both 10 μg/ml; clones 145-2C11 and 37.51 respectively; BioXCell). Th1 conditions comprised recombinant human IL-2 (20 ng/ml; R&D Systems) and mouse IL-12 (20 ng/ml; eBioscience, San Diego, CA, USA), and anti-IL-4 (10 μg/ml; BioXCell).
Extracted molecule genomic DNA
Extraction protocol Polarized Th1 cells from WT and T-bet-/- mice were activated for 4 hours with PMA (50 ng/ml) and ionomycin (1 μg/ml), crosslinked with 11% formaldehyde, lysed and sonicated at 24W for 10 x 30 second pulses using a Misonix Sonicator 3000. The resulting whole cell extract was incubated overnight at 4oC with Dynal Protein G beads preincubated with 10ul of purified rabbit anti-T-bet polyclonal antiserum (9856). Beads were washed and bound complexes eluted and crosslinks reversed by heating at 65oC. IP and input DNA were then purified by treatment with RNAseA, proteinase K and phenol:chloroform extraction. Libraries were constructed from IP and input DNA by standard Illumina protocols, except that DNA in the range 150-350bp was gel-purified after PCR-amplification. The library was quantified using an Agilent bioanalyzer and subjected to 35bp single-end read sequencing with an Illumina Genome Analyzer IIx.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer IIx
Description Chromatin IP against T-bet in Th1 cells
Data processing Initial processing was performed with the CASAVA pipeline. Reads were aligned to the Mouse NCBI37/mm9 reference genome with ELAND, background corrected using whole cell extract data, and converted to tags per million total reads. Significant peaks were identified with MACS using a p-value threshold of 10-6.
Genome_build: mm9
Supplementary_files_format_and_content: bed: alignment (bed format)
Supplementary_files_format_and_content: peaks.txt: MACS peaks (pvalue cut-off <= 1e-6)
Supplementary_files_format_and_content: bw: bigwig
Submission date Sep 05, 2012
Last update date May 15, 2019
Contact name Aditi Kanhere
Organization name University of Liverpool
Street address Institute of Systems, Molecular and Integrative Biology
City Liverpool
ZIP/Postal code L69 3GE
Country United Kingdom
Platform ID GPL11002
Series (1)
GSE40623 Genome-wide regulatory analysis reveals T-bet controls Th17 lineage differentiation through direct suppression of IRF4
SRA SRX184329
BioSample SAMN01163206

Supplementary file Size Download File type/resource
GSM998272_Th1_Tbet.bed.gz 181.6 Mb (ftp)(http) BED 236.8 Mb (ftp)(http) BW
GSM998272_Th1_Tbet_06_peaks.txt.gz 357.2 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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