ARI gene groups (ARI downregulated genes, those highly expressed in BA17 and BA39/40 relative to other regions in controls; ARI upregulated genes, those expressed at low level in BA17 and BA39/40 relative to other regions in controls) were created through taking the union (without duplicates) across all ten identified ASD-attenuated regional comparisons, and sorting genes into the two groups based on gene-expression profiles across regions. The details of this process are described in the Supplementary Methods, along with functional annotation procedures.
Standard workflows using WGCNA17 were followed as previously described in Parikshak et al.5 and Gandal et al.1 (with minor modifications) to identify gene and transcript co-expression modules. Details regarding network formation, module identification, and module functional characterization are described in the Supplementary Methods.
Frozen brain samples were placed on dry ice in a dehydrated dissection chamber to reduce degradation effects from sample thawing and/or humidity. Approximately 50 mg of cortex was sectioned, ensuring specific grey matter–white matter boundary. The tissue section was homogenized in RNase-free conditions with a light detergent briefly on ice using a dounce homogenizer, filtered through a 40-μM filter and centrifuged at 1,000 g for 8 min at 4 °C. The pelleted nuclei were then filtered through a two-part micro gradient (30%/50%) for 20 min at 4 °C. Clean nuclei were pelleted away from debris. The nuclei were washed two more times with PBS/1%BSA/RNase and spun down at 500 g for 5 min. Cells were inspected for quality (shape, colour and membrane integrity) and counted on a Countess II instrument. They were then loaded onto the 10X Genomics platform to isolate single nuclei and generate libraries for RNA sequencing on the NovaS4 or NovaS2 Illumina machines.
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