|
| Status |
Public on Mar 13, 2014 |
| Title |
MLL-AF9 AML KD #2 72hrs |
| Sample type |
RNA |
| |
|
| Source name |
AML cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: MLL-AF9 AML cells
genotype/variation: Pip4k2a knockdown (shRNA #2)
time: 72hrs
|
| Treatment protocol |
N/A
|
| Growth protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using QIAshredder spin columns and an RNAeasy Plus Micro Kit (Qiagen, UK).
|
| Label |
biotin
|
| Label protocol |
RNA quality was confirmed using an Agilent Bioanalyzer (Agilent Technologies, CA) prior to cDNA amplification of 40ng RNA using the Ovation Pico WTA System V2 (NuGEN, CA), according to manufacturer’s instructions. The cDNA was then fragmented and biotin labelled using an Encore Biotin Module kit (NuGEN), according to manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
Fragmented and biotin-labeled cDNA was hybridized to GeneChip Mouse Gene 1.0 ST Arrays (Affymetrix, CA) according to manufacturer’s instructions.
|
| Scan protocol |
Hybridized arrays were washed using the GeneChip Fluidics Station 450 (Affymetrix) using protocol FS450_0007 and scanned using a GeneChip Scanner 3000 7G (Affymetrix).
|
| Data processing |
Microarray data were processed using an R/Bioconductor package. Background adjustment, quantile normalization and summarization of probe-level intensity were performed using the robust multi-array average algorithm. Gene level expression was calculated in R/Bioconductor as the mean of the expression of the exonic probesets for that gene. Associated files used for processing were: MoGene-1_0-st-v1.r4.pgf, MoGene-1_0-st-v1.r4.clf, MoGene-1_0-st-v1.r4.bgp and MoGene-1_0-st-v1.r4.qcc. Affymetrix probe set IDs were remapped to Ensembl mouse gene build 66. The gene level summaries (log2 RMA signals) of Ensembl-annotated protein coding genes are available from the Series record as a supplementary file. To faciliate comparative analysis, only those mouse genes with annotated human homologs were considered further. Mapping was performed using spreadsheets and webtools available at www.informatics.jax.org/homology.shtml and www.genenames.org.
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| |
|
| Submission date |
Jan 22, 2014 |
| Last update date |
Mar 15, 2014 |
| Contact name |
Tim C Somervaille |
| E-mail(s) |
tim.somervaille@cruk.manchester.ac.uk
|
| Organization name |
Cancer Research UK Manchester Institute
|
| Lab |
Leukaemia Biology Laboratory
|
| Street address |
Wilmslow Road
|
| City |
Manchester |
| State/province |
Lancashire |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10740 |
| Series (1) |
| GSE54309 |
A targeted knockdown screen of genes coding for phosphoinositide modulators identifies PIP4K2A as required for acute myeloid leukemia cell proliferation and survival |
|
