|
| Status |
Public on Sep 18, 2017 |
| Title |
poolD_mouse4 |
| Sample type |
SRA |
| |
|
| Source name |
RN2 cells (MLL-AF9/NRASG12D)
|
| Organism |
synthetic construct |
| Characteristics |
shRNA pool: poolD
timepoint: bone marrow day 14
|
| Treatment protocol |
1 x 106 fully selected AML cells were injected in the tail vein of sublethally irradiated (4.5 Gy, 24h before injection) B6.SJL (CD45.1) female mice 6-8 weeks of age. For shRNA induction, animals were treated with doxycycline in the food (625 mg kg-1, Harlan Laboratories). Leukemic mice were euthanized 14 days after transplantation, at terminal disease stage, by CO2.
|
| Growth protocol |
Pools of 50 shRNA vectors were used to produce virus and transduce AML cells at a low multiplicity of infection to minimize double infections. Cells were treated with 500 μg ml-1 G-418 (Roche Applied Science) from two days after infection until fully selected.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Library preparation was performed as previously described (Knott (2014) Mol Cell).
In brief, genomic DNA was extracted from the pre-injection pool and the bone marrow cell suspensions using the QIAamp Blood DNA Maxi Kit (Qiagen). For each sample, shRNA hairpins were extracted from genomic DNA in 96 separate 25-cycle PCR reactions where 2 mg of input DNA was included in each reaction.
Following this initial PCR, Illumina adapters were added via PCR, and samples were processed on the Illumina MiSeq platform.
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
genomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Screen_DESeq_allPools.xls
|
| Data processing |
Reads were extracted and mapped to the shRNAs of the corresponding pool using bowtie (allowing 0 mismatches). Fasta file for the shRNA also included
To analyze the depletion between the final timepoint and input, DESeq was used with Fit type “local”
Genome_build: NA
Supplementary_files_format_and_content: Screen_DESeq_allPools.xls contains the results of DESeq analysis, mjdelasv_GEO_AMLscreen-shRNAs.fasta contains the shRNAs names and sequence.
|
| |
|
| Submission date |
Nov 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
M Joaquina Delas |
| E-mail(s) |
joaquina.delas@crick.ac.uk
|
| Organization name |
The Francis Crick Institute
|
| Department |
Briscoe Lab
|
| Street address |
1 Midland Road
|
| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17769 |
| Series (2) |
| GSE90065 |
shRNA screen identifies lncRNAs required for acute myeloid leukemia progresion |
| GSE90072 |
lncRNA dependencies in acute myeloid leukemia |
|
| Relations |
| BioSample |
SAMN06040934 |
| SRA |
SRX2360590 |
