Quadruplicate biological replicates were collected from ten cortical regions (4–8 individual layers), four hippocampal subfields, and three layers of the LGN (Table S1). Images of pre- and postlaser microdissection are shown in Figure S1. Microdissected tissue was collected directly into RLT buffer from the RNeasy Micro kit (QIAGEN Inc., Valencia, CA) supplemented with β-mercaptoethanol. Samples were volume adjusted with RLT Buffer to 75μl, vortexed, centrifuged, and frozen at −80°C. RNA was isolated for each brain region following the manufacturer’s directions. RNA samples were eluted in 14μl, and 1μl was run on the Agilent 2100 Bioanalyzer (Agilent
Technologies, Inc., Santa Clara, CA) using the Pico 6000 assay kit. Samples NSC 683864 in vivo were quantitated using the Bioanalyzer concentration output. The average RNA Integrity Number (RIN) of all 225 passed
experimental samples was 6.7. Sample amplification, labeling, and microarray processing were performed by the Rosetta Inpharmatics Gene Expression Laboratory (Seattle, WA). Samples passing RNA QC were amplified and profiled as described (Winrow et al., 2009) with a few modifications. Briefly, samples were amplified and labeled using a custom two cycle version, using two kits of the GeneChip HT One-Cycle cDNA Synthesis Kit from Affymetrix. Five nanograms of total RNA was added to the initial PD98059 in vivo reaction mix together with 250 ng of pBR322 (Invitrogen). As little as 2 ng was used in some cases where tissue was extremely limited. Hybridization was performed in three batches to GeneChip Rhesus Macaque Genome Arrays from Affymetrix containing 52,803 probesets/sequences. To control for batch effects, common RNA pool control samples were amplified and hybridized in each batch (3 replicates per 96-well batch). Profile quality was
assessed using standard Affymetrix quality control metrics as well as by PCA. A total of 8 outliers were identified, and these samples were recollected and hybridized successfully. A total of 258 samples passed sample QC, including 225 experimental samples and 33 control samples. The experimental samples include two male and two female profiles for each region (except three missing samples; Table S1). The data discussed in this publication were deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible Thalidomide through GEO Series accession number GSE31613. Each batch was normalized within itself using RMA (Irizarry et al., 2003), and batch effects were removed by subtracting the difference for each probe between controls of one batch from the controls of each other batch. Following this correction, no correlation with batch was observed among all samples within the four primary principal components which explain approximately 40% of the cumulative variance (Figures S2A and S2B; data not shown). ANOVA, principal component and agglomerative clustering was performed using Matlab2007a.