|
| 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 |
| E-mail(s) |
a.kanhere@liverpool.ac.uk
|
| 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 |
|
| Relations |
| SRA |
SRX184329 |
| BioSample |
SAMN01163206 |
