Ssosybr green supermix

Total RNA (1 μg) was reverse transcribed using iScript cDNA synthesis kit (Bio‐Rad) according to the manufacturer's instructions. Using gene sequences available from NCBI for target genes (http://www.ncbi.nlm.nih.gov/nucleotide), PCR oligonucleotide primers were selected and are reported in Table S15. This was done with the NCBI's Primer‐BLAST tool. Real‐time PCR was performed with StepOnePlus^TM Real‐Time PCR System (Invitrogen) using SSOSYBR Green Supermix (Bio‐Rad). Genes were quantified in triplicates; GAPDH was used as housekeeping gene. Gene expression was calculated using the 2^−ΔΔCt method.

For RNA-Seq validation with real-time PCR, eight samples for condition were analyzed (n = 8). Specifically, four experiments were performed each including two samples of NPCs grown in standard floating conditions for 7 days, two samples of NPCs grown inside the Nichoid for 7 days, and two samples of NPCs grown inside the Nichoid and replated in standard floating condition for 7 more days. Total RNA (500 ng) was reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Using gene sequences available from NCBI for target genes (http://www.ncbi.nlm.nih.gov/nucleotide, 29 May 2020) PCR oligonucleotide primers for target genes were selected and primers are reported in Table S12. This was done with the NCBI’s Primer-BLAST tool. Real-time PCR was performed with StepOnePlus^TM Real-Time PCR System (Thermo Fisher, Waltham, MA, USA) using SSOSYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Genes were quantified in triplicates, GAPDH was used as housekeeping gene for mouse samples. Gene expression was calculated using the 2^−ΔΔCt method.

Total RNA (500 ng) was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. Using gene sequences available from NCBI for target genes http://www.ncbi.nlm.nih.gov/nucleotide, PCR oligonucleotide primers for target genes were selected and primers sequences are reported in Table S1 This was done using the NCBI's Primer-BLAST tool. Real-Time PCR was performed with StepOnePlus^TM Real-Time PCR System (Thermo Fisher) using SSO SYBR Green Supermix (Bio-Rad). Genes were quantified in triplicates and GAPDH was used as housekeeping gene. Gene expression was calculated using the 2^-ΔΔCt method.

Total RNA (1000 ng) was reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Using gene sequences available from NCBI for target genes (http://www.ncbi.nlm.nih.gov/nucleotide (accessed on 29 May 2020)) PCR oligonucleotide primers for target genes were selected and primers are reported in Table S1. This was done with the NCBI’s Primer-BLAST tool. Real-time PCR was performed with StepOnePlusTM Real-Time PCR System (Thermo Fisher, Waltham, MA, USA) using SSOSYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Genes were quantified in triplicates, GAPDH was used as housekeeping gene for mouse samples whereas 18S was used for studies in SH-SY5Y. Gene expression was calculated using the 2^−ΔΔCt method.