RNA silencing of UNC93A and WDR27
In order to investigate the potential effects of a loss of function of UNC93A and WDR27 in four brain cell types (Vascular smooth muscle cells, neurons, and astrocytes), we simultaneously silenced both UNC93A and WDR27 genes using a siRNA approach and performed a high throughput RNA sequencing. Through the differential expression analysis, we observed that VSMCs had the highest numbers of differentially expressed genes (DEG=2231), followed by pericytes(DEG=2091), while astrocytes (DEG=226) and neurons (DEG=191) showed a modest change in gene expression compared with control groups (Fig. 3A), suggesting that mutations promoting a loss of function of the UNC93A and WDR27 genes affect the neurovascular unit of the brain. Pathway analysis of VSMC and pericyte gene signatures identified enrichment for multiple known molecular pathways associated with neurological disorders. The data derived of the RNA silencing technique is in the file "Raw data DEGs".
Steps to reproduce
Total RNA was extracted using a miRNeasy kit (Qiagen, Cat. No 217084) following the manufacturer’s protocol instructions. The BGISEQ platform was used for RNA-seq, generating some 4.28G Gb bases per sample, on average. The average mapping ratio with the reference genome was 97.01%, and the average mapping ratio with gene was 74.05%; 17029 genes were identified. We used HISAT to align the clean reads to the reference genome, and Bowtie2 to align the clean reads to the reference genes. 100 ng of total RNA was used for qPCR, as the starting template for cDNA synthesis. The cDNA was prepared by reverse transcription (RT), and gene expression was analyzed by quantitative PCR (qPCR) on a SYBR green system (Applied Biosystems). Expression results were analyzed using the DDCT method, and GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) was used as a house-keeping gene. Fold changes were calculated as the average relative to the control carotid as the baseline.