|
| Status |
Public on Jul 27, 2019 |
| Title |
CBFB-MYH11_-Dox_Runx1-rep1 |
| Sample type |
SRA |
| |
|
| Source name |
CBFB-MYH11_-Dox_Runx1
|
| Organism |
Mus musculus |
| Characteristics |
cell line: myeloid progenitor 416B cell line
construct: CBFB-MYH11
treatment: None
chip antibody: Runx1 (Abcam, ab23980)
|
| Treatment protocol |
Where indicated, cells were induced with 1 µg/ml Doxycycline for 24 hours before harvesting for ChIP-seq.
|
| Growth protocol |
The mouse myeloid progenitor 416B cell line (PMID: 763330) was co-transfected with: 1) a plasmid containing the tetracycline transcription silencer (tTS), the tetracycline transactivator (rtTA) and blasticidine resistance under the control of a Ef1alpha promoter; 2) a plasmid containing the entire Cbfb-Myh11 (CM) type A cDNA in frame with a F2A element and the mCherry protein under the control of a tetracycline responsive element. As a control, cells were alternatively transfected with a version of the plasmid lacking the CM protein. Transposase PL623 (PMID: 21990348, kindly donated by Pentao Liu, Sanger Institute, Cambridge), was also transiently expressed to promote simultaneous stable integration of previous constructs. Plasmids were transfected into 416B cells by electroporation using a BioRad electroporator (220V, 900 µF). After 24h, 1 µg/ml of blasticidin (InvivoGen) was added for selection. After 14 days, mCherry-negative single cells that did not stain with DAPI (Sigma) were sorted into 96-well plates using a BD Influx sorter. Cells were cultured in the presence of selection for typically 2 weeks. Clonal cultures were then tested for induction and expression levels of CM using 1 µg/ml of Doxycycline. Selected clones were expanded for further experiments. Induction of CM for ChIP experiments was confirmed by measurement of mCherry levels by FACS (BD Fortessa).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei following crosslinking with 1% formaldehyde, and histone-DNA complexes were isolated with antibody.
Sequencing libraries were prepared using the TruSeq Kit (Illumina) for high throughput sequencing on an Illumina HiSeq 2500, according to manufacturer’s instructions, with size selection for fragments of 150-400 bp.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
ChIP-seq reads aligned to mm10 using bowtie2 (version 2.0.6) with extra parameters (-k 2 -N 1).
bigWig density profiles were created with in-house script incorporating UCSC's wigToBigWig utility.
Peaks were called using macs2 (version 2.0.10).
Genome_build: mm10
Supplementary_files_format_and_content: [BigWig] read density profile
Supplementary_files_format_and_content: [BED] peaks file
|
| |
|
| Submission date |
Jul 01, 2016 |
| Last update date |
Jul 27, 2019 |
| Contact name |
Evangelia Diamanti |
| E-mail(s) |
ed347@cam.ac.uk
|
| Phone |
01223 62317
|
| Organization name |
University of Cambridge
|
| Department |
Haematology
|
| Lab |
Gottgens
|
| Street address |
Wellcome Trust / MRC Building, Hills Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0XY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE83956 |
CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation |
|
| Relations |
| SRA |
SRX1892679 |
| BioSample |
SAMN05389666 |
