RNA-seq analysis for 3′-UTR alternative polyadenylation identification

RNA-seq data sets for neuron, microglia, astrocyte, oligodendrocyte, and endothelial cells were downloaded from GEO accession series {"type":"entrez-geo","attrs":{"text":"GSE52564","term_id":"52564"}}GSE52564, and data sets for DRG and isolated axons from {"type":"entrez-geo","attrs":{"text":"GSE51572","term_id":"51572"}}GSE51572. Data sets were aligned and processed using HISAT2, Samtools, and Bamtools. Bam or bedgraph files were loaded into IGV for track visualization. For 3′-UTRs alternative polyadenylation analysis, 3′-UTRs reference was built according to QAPA algorithm with the exception of slight modification for Calm1 3′-UTRs. The original QAPA reference contained 6 Calm1 3′-UTR isoforms, but unconfirmed shorter isoforms were omitted for accurate quantification of only Calm1-S and Calm1-L expressions. Isoform abundance was quantified using Sailfish and QAPA quant algorithms. PAU values from the output file were used for comparison of 3′-UTR expression in each cell type.