|
| Status |
Public on Nov 17, 2019 |
| Title |
CTV-1_PU.1-delPmRNA_FA_FLAG-ChIP_1 |
| Sample type |
SRA |
| |
|
| Source name |
CTV-1_PU.1-delPmRNA_FA_FLAG-ChIP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: CTV-1
cell type: T cell leukemia cell line
chip antibody: FLAG M2 (Sigma, #F3165)
transfected ivt mrna: PU.1_delP
fixation method: FA
preperation batch: 1
|
| Treatment protocol |
Cells were transfected with the indicated in vitro transcribed mRNA transcripts (PU1-IVT-mRNA, PU1mut-IVT-mRNA, PU1-delA-IVT-mRNA, PU1-delQ-IVT-mRNA, PU1-delP-IVT-mRNA, PU1-delAQP-IVT-mRNA) and cultured for 8 hours prior to chromatin extraction
|
| Growth protocol |
CTV-1 and HEP-G2 cells were cultured in RPMI 1640 (Gibco; routinely supplemented with 2 mM L-glutamine (Biochrom), 1 mM sodium pyruvate (Sigma), 50 U/ml penicillin/streptomycin, 0.4x vitamins (Sigma), 1x non-essential amino acids (Sigma), 50 μM b-mercaptoethanol (Gibco)) supplemented with 10% heat inactivated fetal bovine serum; For TALL-1 cells the culture medium was supplemented with 15% heat inactivated fetal bovine serum was used; adherent HEP-G2 cells were detached using Trypsin-EDTA (Gibco)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were preparated from sonicated nuclei and protein/histone-DNA complexes were isolated with antibody.
ChIP construction was essentially done as described (Pham et al. 2012) with slight modifications for BRG1, ETS1 and FLI1 samples, which were prepared using dual crosslinking with 2 mM DSG (Thermo Fisher Scientific) and 1% formaldehyde. Libraries for sequencing were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina according to manufacturer’s instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
Chromatin IP against FLAG-epitope, 8h after mRNA transfection
|
| Data processing |
Raw data were aligned to the human genome (GRCh37/hg19) using bowtie2 (Langmead and Salzberg, 2012) in very sensitive mode, keeping only reads that map to a single unique genomic location for further analysis (MAPQ > 10).
Bigwigs of ChIPseq data sets were generated using HOMER v4.9 (Heinz et al, 2010) and standard settings. For cancer cell lines, we used the Control-FREEC v11.0 program to determine allelic imbalances from low-depth whole genome sequencing data and corrected ChIP-seq read counts accordingly to generate CNV-corrected bigWigs.
ChIP-seq peaks were called using HOMER?s findPeaks program in ??factor?? mode using default parameters (standard) or with -fdr 0.00001 (stringent) to identify focal peaks. Stringent peaks were further filtered for a minimal normalized tag count of 15 tags per peak. All peak sets were filtered by subtracting blacklisted genomic regions 49, and by filtering out regions with a mappability <0.8. The latter was annotated to peak regions from mappability tracks generated with the GEM package using HOMER?s annotatePeaks.pl.
Genome_build: hg19
Supplementary_files_format_and_content: ChIPseq tracks in bigWig format (before and after CNV normalization
Supplementary_files_format_and_content: ChIPseq peaks (called using standard or stringent parameters)
|
| |
|
| Submission date |
Mar 25, 2019 |
| Last update date |
Nov 19, 2019 |
| Contact name |
Michael Rehli |
| E-mail(s) |
michael.rehli@klinik.uni-r.de
|
| Organization name |
University Hospital Regensburg
|
| Department |
Internal Med III
|
| Street address |
F.-J.-Strauss-Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93042 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21290 |
| Series (2) |
| GSE128835 |
The hematopoietic master transcription factor PU.1 requires its interaction with the SWI/SNF remodeler to access chromatin de novo [CTV1_TALL_HELP ChIP-seq] |
| GSE128837 |
The hematopoietic master transcription factor PU.1 requires its interaction with the SWI/SNF remodeler to access chromatin de novo |
|
| Relations |
| BioSample |
SAMN11252816 |
| SRA |
SRX5574491 |
