NALM6 cells were engrated in SCID mice, which were treated with control/PI3Kd until animals reached clinical endpoint.
Extracted molecule
total RNA
Extraction protocol
Mouse femurs were collected and BM cells were aspirated with RPMI1640 containing 10% FBS. Murine spleen tissues were homogenized in RPMI1640 containing 10% FBS. The spine was removed from mice, cut into columns and aspirated with RPMI1640 containing 10% FBS to collect CSF cells. The BM, spleen and CSF cells were passed through 70 μm filters, washed with PBS and treated with ACK lysis buffer.RNA was extracted using RNAeasy kit (Qiagen, Germantown, MD) and total RNA were assessed for quality with Agilent 2100 Bioanalyzer G2939A (Agilent Technologies,Santa Clara, CA) and Nanodrop 8000 spectrophotometer (Thermo Scientific/Nanodrop, Wilmington, DE).
Label
biotin
Label protocol
Affymetrix WT Plus Kit (PN 902280) and GeneChip WT Terminal Labeling Kit (PN 900670)
Hybridization protocol
Hybridization targets were prepared with the Affymetrix WT Plus Kit (Affymetrix, Santa Clara, CA) and Affymetrix GeneChip WT Terminal Labeling Kit (included with Affy WT Plus) from total RNA, hybridized to GeneChip® Human Clariom D arrays in Affymetrix GeneChip® hybridization oven 645, washed in Affymetrix GeneChip® Fluidics Station 450 and scanned with Affymetrix GeneChip® Scanner 7G according to standard Affymetrix GeneChip® Hybridization, Wash, and Stain protocols. (Affymetrix, Santa Clara,CA).
Scan protocol
Affymetrix scanning protocol according to manufacturer, Affymetrix GeneChip Command Console Scan Control 3.2.3.1515 and Expression Console version 1.2.0.20
Description
NALM6 human ALL cells isolated from SCID mice after control/PI3Kd treatment
Data processing
Total RNA isolated from BM and CSF was sent for gene expression profiling using the Clariom D Human microarrays (Affymetrix, Santa Clara, USA) at the Duke Center for Genomic and Computational Biology. Raw cell intensity data was imported in to Expression Console (Affymetrix, Santa Clara, USA) and BM arrays (vehicle and GS-649443 samples), and CSF arrays (vehicle and GS-649443) were normalized using the RMA algorithm. Gene level differential expression analysis was performed using the Transcript Analysis Console (v3.1.0.5: Affymetrix, Santa Clara, USA) and ANOVA statistical analysis performed on the BM and CSF arrays separately. Transcripts identified to be differentially regulated in the BM, or CSF samples by ±1.5-fold with a p-value <0.05 were carried forward for pathway analysis.
