RNA-seq Analysis of Neural Cell Types

Total RNA samples were extracted from NPCs, neurons, and astrocytes using a Qiagen RNA Isolation Kit (Qiagen, Hilden, Germany) with DNAase I treatment (Qiagen). mRNA was isolated from total RNA using a Dynabeads mRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). mRNA samples (200 ng) were used to construct RNA libraries using MGIEasy RNA Library Prep kit (MGI, Shenzhen, China) and sequenced with MGISEQ-2000 sequencing platform. We used Salmon software [89 (link)] with default parameters to map RNA-seq reads to the human transcriptome release 37 from GENCODE (Frankish et al., 2019). The same software was used to count the number of RNA reads mapped to each reference transcript. The transcript-level quantification results were converted to the gene-level quantities with tximport [90 (link)] R package. Finally, DESeq2 [91 (link)] R package was used to normalize the gene-level quantities and plot PCA and heatmap from the normalization results.