RNA Isolation and qRT-PCR Analysis of Neuronal Transcripts

iPSNs were harvested in 1 × DPBS with calcium and magnesium and pelleted using a microcentrifuge. 350 μL RLT Buffer was added to iPSN pellets and RNA was isolated using the RNeasy kit (QIAGEN). RNA concentrations were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). For detection of G₄C₂ and G₂C₄ repeat containing transcripts, 1 μg RNA was used for cDNA synthesis using gene specific primers and the Superscript IV First-Strand cDNA Synthesis System (Thermo Fisher Scientific). For detection of TDP-43 mRNA targets, 1 μg RNA was used for cDNA synthesis using random hexamers and the Superscript IV First-Strand cDNA Synthesis System (Thermo Fisher Scientific). All qRT-qPCR reactions were carried out using SYBR Green Master Mix or TaqMan Gene Expression Master Mix (Thermo Fisher) and an Applied Biosystems QuantStudio 3 (Applied Biosystems). Previously described primer/probe sets (see Supplemental Table 2 for sequences) [24 (link)] were used to detect G₄C₂ and G₂C₄ repeat containing transcripts. Previously described primer sets (see Supplemental Table 2 for sequences) [30 (link), 32 (link), 40 (link), 43 (link)] were used to detect truncated STMN2 and cryptic exon containing mRNA transcripts. TaqMan Gene Expression Assays (see Supplemental Table 2 for probe information) were used to detect mRNA targets. GAPDH was used for normalization of gene expression.