Twenty milligrams of the frontal lobe of P14 mice of WT, Het, KO and treated KO (n=6 per genotype) was immediately removed after euthanizing the mice with isoflurane. RNA extraction was performed following the manufacturer’s instructions (RNeasy mini kit, Qiagen, Hilden, Germany) and DNase treatment (Roche, Basel, Switzerland) was applied.
Label
biotin
Label protocol
RNA was transcribed to double-stranded cRNA using MessageAmp Premier RNA Amplification Kit (Life Technologies, Carlsbad, CA) with an oligo(dT) primer following the manufacturer’s instructions. Following fragmentation, 11 µg of biotin-labeled cRNA were hybridized for 16 hr at 45°C on Affymetrix Mouse Genome 430 Plus 2.0 Arrays (Affymetrix, Santa Clara, CA). GeneChips were washed and stained in the Affymetrix Fluidics Station 450 (Affymetrix), then scanned with the Affymetrix GeneChip Scanner 3000 G7 (Affymetrix).
Hybridization protocol
Following fragmentation, 11 µg of biotin-labeled cRNA were hybridized for 16 hr at 45°C on Affymetrix Mouse Genome 430 Plus 2.0 Arrays (Affymetrix, Santa Clara, CA). GeneChips were washed and stained in the Affymetrix Fluidics Station 450 (Affymetrix), then scanned with the Affymetrix GeneChip Scanner 3000 G7 (Affymetrix).
Scan protocol
GeneChips were washed and stained in the Affymetrix Fluidics Station 450 (Affymetrix), then scanned with the Affymetrix GeneChip Scanner 3000 G7 (Affymetrix).
Description
Array hybridization was done by the UCLA Clinical Microarray Core using their protocols.
Data processing
Raw fluorescence data was stored as .CEL.gz files was loaded into R as an AffyBatch object (using the affy package in R). This object was then processed using Robust Multi-Array Average (RMA) normalization, also part of the affy package, which removed background ‘noise’ and converted data to a log2 scale. ComBat from the sva R package was then applied to the normalized data to adjust for batch effects. Affymetrix probes were summarized and re-annotated with Ensembl gene names from the Ensembl July 2015 archive (Ensembl version 81). Finally, the dataset was filtered so that genes with a standard deviation in the 5th quantile or lower were removed.
