|
| Status |
Public on Dec 23, 2019 |
| Title |
in vivo BB518 DMSO Rep2 [TS92_4_1-7] |
| Sample type |
SRA |
| |
|
| Source name |
in vivo BB518 DMSO
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary MLL-AML patient cells
host mouse strain: NSG
treatment: DMSO (vehicle) for 24 hours
|
| Treatment protocol |
5-10x10^6 million cells were injected in sub-lethally irradiated (1 Gy) female NSG mice (6-8 weeks, Envigo) via the tail vein. Tail bleeds were performed 8 weeks after transplantation and blood engraftment levels and lineage contributions determined by flow cytometry analysis of leukocyte populations following ammonium chloride lysis of erythrocytes. When transplanted mice exhibited signs of ill health they were euthanized and leukaemia cells from BM and spleen cryopreserved for later use. After passaging of MLL-AML cells through NSG mice twice, 1x10^5 cells were injected in sub-lethally irradiated (50 cGy) female NSG mice (6-8 weeks, Envigo) via the tail vein. 34 days after transplantation, mice were divided in to 4 groups treated via oral gavage with either vehicle, RAD001 (5mg/kg), OG98 (3mg/kg), or RAD001 (5mg/kg) + OG98 (3mg/kg) for 5 days.
|
| Growth protocol |
Primary MLL-AML patient samples were recovered by thawing cells directly into PBS supplemented with 20% human serum albumin, 4µg/ml DNase1, 2.5mM MgCl2 and 16.4mM trisodium citrate (“DAMP solution”) followed by CD3+ cell depletion (Beads) using the POSSELD program of an AutoMACS Pro device (both from Miltenyi Biotec) according to the manufacturer’s instructions. Cells were placed over night in StemSpanTM at a density of 2.5x104 /ml supplemented with 100ng/ml each of FL, IL-6, TPO and SCF.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from DMSO or OG86-treated THP1 AML cells using QIAshredder spin columns and an RNeasy Plus Micro Kit (Qiagen, Manchester, UK) and its quality confirmed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
TS92_4_1-7_S4
|
| Data processing |
Paired-end reads were mapped to the human genome (GRCh38 Gencode release 25) with STAR version 2.4.2a with the settings --outFilterMultimapNmax 20, --outFilterType BySJout, --alignSJoverhangMin 8 and --quantMode GeneCounts using the matching feature annotation GTF file for GRCh38 Gencode release 25. Differential gene expression analysis was performed using DESeq2 version 1.20.0.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include raw read count values per gene for each sample
|
| |
|
| Submission date |
Feb 13, 2019 |
| Last update date |
Feb 05, 2020 |
| Contact name |
Tim C Somervaille |
| E-mail(s) |
tim.somervaille@cruk.manchester.ac.uk
|
| Organization name |
Cancer Research UK
|
| Department |
Manchester Institute
|
| Lab |
Leukaemia Biology
|
| Street address |
Wilmslow Rd
|
| City |
Manchester |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE126486 |
Genome-wide CRISPR-Cas9 screen identifies druggable synthetic lethality between LSD1 and MTORC1 in MLL-translocated AML |
|
| Relations |
| BioSample |
SAMN10924205 |
| SRA |
SRX5371470 |
