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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 14, 2020 |
| Title |
Runx1Evi1_plusDox_H3K27ac_976 |
| Sample type |
SRA |
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| Source name |
A2lox.cre mESC
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| Organism |
Mus musculus |
| Characteristics |
cell type: A2lox ESCs
treatment: Runx1Evi1_plusDox
target: H3K27ac
chip antibody: Abcam ab4729
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| Treatment protocol |
ESCs were differentiated as previously described (Gilmour et al, 2014; Regha et al., 2015) with the following modifications. FLK1+ cells were purified by magnetic cells sorting, using biotin-conjugated CD309 antibody (eBioscience), anti-biotin microbeads (Miltenyi Biotec) and LS columns (Miltenyi Biotec) following culture of embryoid bodies for between 3.25 and 3.75 days (cell line dependent). These FLK1+ cells were then cultured in gelatin-coated flasks – 1.2-1.4x10e6 cells in a T150 flask to form the blast culture. After 1-2 days (cell line dependent), 0.1-0.5 µg/ml doxycycline was added as appropriate and cells were cultured in the same media for a further 18 hours prior to sorting for HE and Progs. Cell populations were identified and sorted on day 2-3 of blast culture based on surface markers. Cells were stained with KIT-APC (BD pharmingen), Tie2-PE (eBioscience) and CD41-PE-Cy7 (eBioscience) and analyzed on a Cyan ADP flow cytometer (Beckman Coulter) with data analysis using FlowJo, or sorted on a FACS Aria cell sorter (BD Biosciences).
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| Growth protocol |
Generation of RUNX1-ETO and RUNX1-EVI1 containing ESCs was previously described (Kellaway et al., 2020; Regha et al., 2015). R201Q and R204X plasmids were generated by site-directed mutagenesis on wild type human RUNX1c, and N-terminal HA tags added using the following primers: R201Q forward 5’-CAGTGGATGGGCCCCAAGAACCTCGAAGAC-3’, reverse 5’-GTCTTCGAGGTTCTTGGGGCCCATCCACTG-3’ and R204X forward 5’-CCCCCTCGAGCCACCATG-3’, reverse 5’-GCCGATGATATCTCAAGGTTCTCG-3’. A2lox ESCs (a gift from Michael Kyba) were transduced with 20 µg of each plasmid using the 4D-Nucleofector (Lonza), mouse ES program and P3 primary cell kit. Note, R201Q, R204X and RUNX1-ETO all included N-terminal HA-tags.Individual colonies were expanded and maintained on mouse embryonic feeder cells in ES cell medium, comprising DMEM (Sigma D6546), 15% FCS (Sigma ES-009), 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 1mM L-glutamine, 0.15 mM monothioglycerol, 1x non-essential amino acids and 103 U/ml leukaemia inhibitory factor (ESGRO, Millipore) following 7 days of 300 µg/ml neomycin selection.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as previously described (Obier et al, 2016) with the following modifications. For H3K27ac only KIT+ Progs were crosslinked only in 1% formaldehyde (single crosslinking, as opposed to double crosslinking for RUNX1). For single crosslinked cells nuclei were sonicated for 4 cycles of 30s on/30s off using a Picoruptor (Diagenode). Immunoprecipitation was carried out overnight at 4°C using 2 µg of RUNX1 antibody (ab23980, Abcam), or for four hours at 4°C using 2 µg of H3K27ac antibody (??) coupled to 15 µg Dynabeads Protein G (Invitrogen) per 2 x 106 cells. DNA from 2-3 immunoprecipitations was pooled for RUNX1, but just 1 immunoprecipitation for H3K27ac, and extracted using Ampure beads (Beckman Coulter).
ChIP libraries were generated using the KAPA hyper prep kit, libraries were size selected to obtain fragments between 150 and 450 bp
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw single-end reads were processed with Trimmomatic v0.32.
The processed reads were aligned to the mouse genome (version mm10) using Bowtie2 v2.2.3 with the option --very-sensitive-local
Peaks were identified using MACS v2.1.1 with the options -g mm --keep-dup all -B --trackline
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph files of sequence read pileups generated by MACS2
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| Submission date |
Jul 17, 2020 |
| Last update date |
Dec 15, 2020 |
| 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 |
GPL19057 |
| Series (2) |
| GSE154621 |
Different mutant RUNX1 oncoprotein classes program alternate hematopoietic differentiation trajectories [ChIP-Seq] |
| GSE154623 |
Different mutant RUNX1 oncoprotein classes program alternate hematopoietic differentiation trajectories |
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| Relations |
| BioSample |
SAMN15567490 |
| SRA |
SRX8754240 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4675981_Runx1Evi1_plusDox_H3K27ac_976_treat_pileup.bedgraph.gz |
174.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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