Rett syndrome-causing MECP2 mutations severely impact key cellular and molecular features of human astrocytes

Purpose: The goal of this RNA sequencing (RNA-seq) study is to identify aberrations in the astrocyte transcriptional landscape caused by R270X mutation in MECP2. Methods: mRNA-seq analysis was performed on total RNA extracted from human embryonic stem cell (ESCs)-derived wild-type (WT) and MECP2-R270X mutant astrocytes. The R270X mutation was inserted into ESCs (line H1) via CRISPR/Cas9 technology. Samples were generated in triplicates, sequenced by Illumina NovaSeq 6000 Sequencing System. Raw reads were first trimmed for 10 bases at the 5’end to remove reads with biased nucleotide (ACGT) distribution. Trimmed reads were then aligned to the Homo sapiens genome (GRCh38p12, GENCODE, primary assembly), using STAR aligner. Differential gene expression (DEG) analyses on the read counts were performed using DESeq2. Genes with sum of read counts across all samples with less 10 were filtered out from analysis. Results: Our RNA-seq analysis showed that 1,621 genes were dysregulated in mutant astrocytes (fold-change >1.5 or <2/3; padj < 0.05, average reads count >10 in at least one genotype) The mutant samples and their isogenic WT control samples were generated in triplicates.