Quantitative RT-PCR Analysis of Gene Expression

Total RNA was extracted from cells using RNeasy Mini Kit (Qiagen). cDNA was made by reverse-transcribing 1 μg of total RNA using MuLV Reverse Transcriptase and Oligo (dT) primers (Applied Biosystems). qRT-PCR was performed with a Bio-Rad CFX Detection System (Bio-Rad) and expression of target genes was measured using Power SYBR green PCR kit (Applied Biosystems). Samples were amplified in triplicate and relative gene expression was analyzed using Bio-Rad CFX manager software and normalized to 18S RNA. Primer sequences were used to measure the expression of reprogramming transcription factors, stem cell and neural lineage markers were previously reported by us.^{21 (link)} Primer sequences used in this study were obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/) and are listed in Supplementary Tables 1 and 2.