Rnaqueous micro total rna kit

For O9-1 cells, RNA was isolated from cells using RNAqueous-Micro Total RNA Isolation Kit (Ambion, AM1931) following the manufacturer's protocol. 1 µg RNA was reverse transcribed into cDNAs using High-Capacity RNA-to-cDNA Kit (Applied Biosystems, 4387406) following the manufacturer's instructions. PowerUp SYBR Green Master Mix (Applied Biosystems, A25742) was used to run qRT-PCR. Serial dilutions (10 ng, 5 ng, 2.5 ng and 1.25 ng) of cDNA were used to test qRT-PCR efficiency. All qRT-PCR experiments were performed with at least three biological replicates, and each contained three technical replicates. Primers used for qRT-PCR are listed in Table S4.
For hiPSC-derived NCCs, RNA was extracted from pelleted cells and DNase treated (RNAqueous-Micro Total RNA kit, Invitrogen). cDNA was synthesized with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). qRT-PCR was performed in triplicate using PowerUp SYBR Green Master Mix (Applied Biosystems, A25742) on Applied Biosystems QuantStudio 6 Flex Real-Time PCR System. All genes were normalized to Gapdh expression. NCCs at P3 were used for qPCR controls. Primers used for qRT-PCR are listed in Table S4.

Total RNA was extracted from in vitro tissue using the MagMAX mirVana Total RNA kit (Applied Biosystems) and from fetal tissue using the RNAqueous-Micro Total RNA kit (Invitrogen). RNA (0.1–1 μg) was reverse transcribed with Superscript IV VILO reverse transcriptase (Invitrogen) and treated with ezDNase enzyme (Invitrogen). Real-time quantitative PCR was performed on a ViiA 7 Real-Time PCR System with OptiFlex Optics System (Applied Biosystems) using PowerUp SYBR Green PCR kit (Applied Biosystems). Genomic DNA standards were used to evaluate the efficiency of the PCR and calculate the copy number of each gene relative to the expression of the gene encoding TATA-box binding protein (TBP). All data represent three biological replicates (independent experiments or fetal donor specimens) or more as indicated. Student’s t-test was used to evaluate statistical significance, as indicated. Oligonucleotides are provided in Supplementary file 6.