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Pipeline publication

[…] etwork and 0.7 for TF network resulted in a co-expression network with 753 (DEG) and 431 (TF) nodes. The calculated gene-gene pairs, along with their Pearson correlation coefficients, were exported from BioLayout3D and imported into Cytoscape () using force-directed layout for visualization. Group FC values (based on the comparison of each condition with the mean of all conditions) were mapped onto the network for each condition individually. To identify commonalities and differences between microglia at different stages based on co-expression network analysis, we colored genes based on their group FCs > 1.5 or < −1.5., Raw reads were first aligned to mouse reference genome MM10 using STAR aligner. Read count per gene was then calculated using the FeatureCount program and gencode gene annotation version M9. Count per million read (CPM) values were calculated using the edgeR package. DEGs showing adjusted p values less than 0.05, fold change greater than 1.5, and a minimum of 25 average CPM were identified using edgeR package., Weighted gene co-expression network analysis was performed on normalized expression data using the R package WGCNA v1.51 () following the standard workflow. For computational efficiency, genes were filtered according to the subsequent procedure: First, for each comparison, the expression table was subsetted by all samples associated with the respective comparison and the 1000 most variably expressed genes between those samples were determined. The resulting 11 sets of 1000 most variably expressed genes were then united accounting for 2395 genes representing the relevant variance within the dataset. To obtain a signed network fulfilling the scale free topology, the soft-thresholding power parameter was set to 20. Co-expression modules were defined using a minimum module size of 30 genes and by mergi […]

Pipeline specifications

Software tools STAR, edgeR, WGCNA