|
| Status |
Public on Mar 30, 2022 |
| Title |
EL4_GATA3_Dox_Tbet_DOX_10_HA-GATA3_ChIP |
| Sample type |
SRA |
| |
|
| Source name |
EL4 cell
|
| Organism |
Mus musculus |
| Characteristics |
cell line: EL4 cells
ectopically expressed protein: HA-GATA3+ Dox-inducible FLAG-T-bet
treatment: PMA+ionomycin; 10ug/ml Dox
antibody: anti-HA, 3F10 Roche
|
| Treatment protocol |
Cells were treated with 0-10ug/ml doxycycline for 48 hour prior to harvesting. Cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 μM) for 4 hours prior to crosslinking. Cells were crosslinked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 20 minutes at room temperature before the reaction was quenched by addition of glycine as described. Cells were rinsed twice with 1xPBS and flash frozen in liquid nitrogen.
|
| Growth protocol |
EL4 GFP+HA-GATA3 cells were transduced with retrovirus encoding a doxycycline-inducible FLAG-T-bet construct and selected with G418. 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 |
Cells were lysed with non-ionic detergent, the nuclei washed and then lysed with ionic detergent. Cells were sonicated on ice to solubilize and shear crosslinked DNA (27W for 10x 30 second pulses using a Misonix Sonicator 3000). The resulting whole cell lysate was cleared by centrifugation and then incubated overnight at 4°C with 50 µl of Protein G magnetic Dynabeads that had been pre-incubated with 5 µg of purified antibody (anti-FLAG, M2 Sigma; anti-HA, 3F10 Roche; anti-H3K27ac, ab4729 Abcam). Beads were washed 6 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65°C with rocking for 2 hrs and crosslinks then reversed in IP and input DNA by incubation at 65°C for 6 hrs. IP and input DNA were then purified by treatment with RNase A, proteinase K and isolated with KAPA Pure beads.
Libraries were constructed from ChIP DNA by standard Illumina protocols, except that DNA in the range 150-350 bp was gel-purified after PCR-amplification. The libraries were quantified using a Qubit fluorometer and Agilent bioanalyzer, pooled and subjected to 75 bp single-end sequencing with a NextSeq 550 sequencer
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
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 with default settings
Genome_build: mm10
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
Feb 10, 2022 |
| 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 |
GPL21626 |
| Series (1) |
| GSE196508 |
The TH1 cell lineage-determining transcription factor T-bet supresses TH2 gene expression by redistributing GATA3 away from TH2 genes [ChIP-seq series 2] |
|
| Relations |
| BioSample |
SAMN25845562 |
| SRA |
SRX14130418 |
