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| Status |
Public on Nov 04, 2022 |
| Title |
KO52_dnCEBP_ATAC_rep2 |
| Sample type |
SRA |
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| Source name |
KO52
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| Organism |
Homo sapiens |
| Characteristics |
cell line: KO52
genotype: CEBPA double mutant
treatment: dnCEBP
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| Treatment protocol |
The doxycycline inducible pCW57.1-dnC/EBP plasmid was generated using Gateway Gene Cloning (Thermo Fisher Scientific), according to manufacturer’s instructions. The CMV500 A- C/EBP plasmid containing the dnC/EBP insert was a gift from Charles Vinson, National Cancer Institute, Bethesda, USA 1. dnC/EBP cDNA was PCR amplified (with Phusion DNA polymerase (New England Biolabs) using primers containing SalII and NotI restriction enzymes overhangs and cloned into pENTR-IRES-GFP (a modification of the Invitrogen pENTR plasmid constructed by Benjamin Edginton-White, University of Birmingham, UK) digested with 30 units of SalI-HF (New England Biolabs), 30 units of NotI-HF (New England Biolabs). The dnC/EBP insert was then transferred into the Tet-on pCW57.1 backbone (Addgene plasmid #41393) by using the Gateway Gene Cloning System. The pCW57.1 containing the dnFOS insert was generated by Dr Sandeep Potluri (University of Birmingham) following the same protocol. Lentiviral particles containing pCW57.1-dnC/EBP / dnFOS or the empty vector were generated in HEK293T cells using calcium-phosphate transfection. Cells were cultured in DMEM supplemented with 10% foetal calf serum (FCS), 5% Penicillin-Streptomycin (Pen-Strep), 5% L-Glutamine (L-Glu) and 5% Sodium Pyruvate and seeded to achieve about 30-40% confluency at the time of transfection. 12 μg of pCW57.1-dnC/EBP or empty vector were transfected with 0.6 ug of REV, 0.6 ug of GAG/POL, 1.2 ug of VSV-G and 0.6 ug of TAT packaging plasmids. Viral supernatant was harvested at 24, 36, 48, 60 and 72h and concentrated with Centricon Plus- 70 columns (Merk-Millipore) using manufacturer’s instructions. Concentrated viral particles were filtered through 0.45 um filter and stored at -80°C or immediately used to infect recipient cells. To carry out the spin infection, 1 x 10^6 Kasumi-1 or KO52 cells were plated in 17 6-well plates with the viral supernatant and 8 ug/mL of polybrene, and centrifuged for 1h 30m at 1500 x g at 32°C. After infection cells were incubated at 37 °C for 12 hours and then resuspended in fresh media. Few days after infection, cells were cultured in medium supplemented with puromycin 1.5 ug/mL and 2ug/mL for 5 days, allowing the selection of cells positive for pCW57.1-dnC/EBP. Single clones were then isolated from the bulk Kasumi-1 population, expanded and tested for leakiness and induction of dnC/EBP measuring both GFP and FLAG expression through both FACS and RT-qPCR. Despite several attempts, it was not possible to expand KO52 single cell clones with either vector and the bulk population was used for downstream experiments. Dominant negative peptides expression was induced by adding 2 μg/mL of doxycycline to culture medium.
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| Growth protocol |
KO52 were cultured in Alpha MEM (Lonza) supplemented with 10% FCS (Gibco), 5% Pen-Strep, 5% L-Glu.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC libraries were generated following the Omni-ATAC protocol from Corces et al, 2017 (PMID:28846090)
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Sequencing adapters and low-quality reads and were removed using Trimmomatic
Reads were then mapped to the human genome version hg38 using Bowtie v2.2.3
PCR duplicated reads were removed using the MarkDuplicates funtion Picard tools
Open chromatin regions (peaks) were identified using MACS2 and bedGraph files were generated by including the options -B --trackline
Assembly: hg38
Supplementary files format and content: bedGraph files of read pileups generated by MACS2
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| Submission date |
Aug 11, 2022 |
| Last update date |
Nov 04, 2022 |
| Contact name |
Peter Keane |
| E-mail(s) |
p.keane@bham.ac.uk
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| Organization name |
University of Birmingham
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| Department |
Institute for Cancer and Genomic Sciences
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| Street address |
Vincent Drive
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| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE211041 |
Identification and interrogation of the gene regulatory network of CEBPA double mutant Acute Myeloid Leukaemia [KO52 ATAC-seq] |
| GSE211095 |
Identification and interrogation of the gene regulatory network of CEBPA double mutant Acute Myeloid Leukaemia |
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| Relations |
| BioSample |
SAMN30265320 |
| SRA |
SRX17033222 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6448276_KO52_dnCEBP_ATAC_rep2_treat_pileup.bedGraph.gz |
149.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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