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| Status |
Public on May 01, 2024 |
| Title |
D1091-11_ITD-18_RASi_Rep2 |
| Sample type |
SRA |
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| Source name |
CD34+ primary AML cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD34+ primary AML cells
genotype: FLT3-ITD+
treatment: RASi
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| Growth protocol |
Cells were cukltered at 0.3-0.5 x 106 cells/ml on hMSC feeders in alpha-MEM (Lonza) supplemented with 12.5 % fetal calf serum, 12.5 % horse serum, 100 U/ml penicillin/streptomycin, 2 mM L-Glutamine (all Gibco), 1 μM hydrocortisone (Merck) and 57.2 μM β-mercaptoethanol (Merck), 20 ng/ml IL-3, G-CSF and TPO (Pepro Tech). Cells were cultured on hMSC feeders for 7 days prior to experiments. MV4-11 (DMSZ, AC102) and MOLM14 (DSMZ, ACC 777) were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 2mM L-glutamine and 100 u/ml senicillin/streptomycin (all Gibco) at 37 ˚C with 5% CO2. Human embryonic kidney 293T (HEK293T) cells were cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.11 mg ml–1 sodium pyruvate.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were treated with inhibitors in the presence or absence of IL-3 for 24 h prior to harvest. Omni ATAC-seq was performed as in Corces et al. Briefly, cells were washed in ATAC resuspension buffer (RSB) (10mM Tris-HCl pH7.5, 10mM NaCl and 3mM MgCl2) and then lysed for 3 minutes on ice in RSB buffer with 0.1% NP-40, 0.1% Tween-20. Then the cells were washed with 1ml of ATAC wash buffer consisting of RSB with 0.1% Tween-20. Then the nuclear pellet was resuspended in ATAC transposition buffer consisting of 25μl TD buffer and a concentration of Tn5 transposase enzyme (Illumina) related to the number of input cells, 16.5 μl PBS, 5 μl water, 0.1% tween-20 and 0.01% digitonin and then incubated on a thermomixer at 37°C for 30 minutes. The transposed DNA was then amplified by PCR amplification up to ¼ of maximum amplification, as assessed by a qPCR side reaction.
The library was purified using a QIAquick PCR cleanup kit (QIAGEN) followed by ampure (Beckman Coulter) and analysed on a Next Seq 2000 75 using a NextSeq 500/550 High output kit.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Single-end reads from ATAC-Seq experiments were trimmed with Trimmomatic and aligned to the human genome (version hg38) using Bowtie2 with the --very-sensitrive-local parameter.
Potential PCR duplicates were identified and removed from alignments using the MarkDuplicates function in Picard.
Peak calling was then carried out using MACS2 with the options --nomodel --shift -100 --extsize 200 -B --trackline.
Assembly: hg38
Supplementary files format and content: BedGraph files of read pileups from MACS2
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| Submission date |
Aug 24, 2023 |
| Last update date |
May 01, 2024 |
| 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 |
GPL30173 |
| Series (2) |
| GSE241646 |
Pharmaceutical inhibition of RAS overcomes cytokine mediated resistance to FLT3 inhibition in FLT3-ITD+ AML [ATAC-seq] |
| GSE241650 |
Pharmaceutical inhibition of RAS overcomes cytokine mediated resistance to FLT3 inhibition in FLT3-ITD+ AML |
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| Relations |
| BioSample |
SAMN37144963 |
| SRA |
SRX21479921 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7732644_D1091-11_ITD-18_RASi_Rep2_treat_pileup.bedgraph.gz |
101.1 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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