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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 14, 2020 |
| Title |
Runx1Eto_PlusDox_HP_Rep2 |
| 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: Runx1Eto_PlusDox
sorted cells: HP
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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 |
total RNA |
| Extraction protocol |
RNA was isolated from sorted cells using either the NucleoSpin RNA kit (Macherey-Nagel) or Trizol reagent (Thermo Fisher Scientific).
RNA-seq libraries were prepared from two biological replicates using the True-Seq stranded total RNA kit (Illumina) and sequenced paired-end in a pool of 12 indexed libraries using a Next-Seq 500/550 high output kit v2 150 cycles (Illumina)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw paired-end sequencing data were processed to remove low quality sequences and adaptors with Trimmomatic v0.32
The processed reads were then aligned to the mouse genome (version mm10) using Hisat2 v2.1.0 with default parameters
Gene expression was measured as raw counts with FeatureCounts using gene models from RefSeq as the reference transcriptome
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited file of read counts
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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) |
| GSE154622 |
Different mutant RUNX1 oncoprotein classes program alternate hematopoietic differentiation trajectories [RNA-Seq] |
| GSE154623 |
Different mutant RUNX1 oncoprotein classes program alternate hematopoietic differentiation trajectories |
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| Relations |
| BioSample |
SAMN15567518 |
| SRA |
SRX8754264 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4676005_Runx1Eto_PlusDox_HP_Rep2_rawCounts.tsv.gz |
132.5 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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