Preamp and reverse transcription master mix

Total RNA was isolated from FAC‐sorted GFP positive cells from Hes5::GFP (E11.5, E15.5 and E18.5) and Tbr2::GP (E13.5 BPs, E15.5 BPs and E15.5 NBNs) transgenic embryos using TRIzol reagent. Independent biological replicates were generated for RT‐qPCR validation. Samples were analyzed for their integrity and concentration using Agilent 2100 Bioanalyzer and Quant‐IT RiboGreen RNA Assay Kit. DNase treatment was done using Roche DNase kit and cDNA prepared using the PreAmp and Reverse Transcription Master Mix from Fluidigm. Deltagene Assay primers (Fluidigm) and EvaGreen (BioRad) were used for real‐time qPCR. Gene expression was assayed using Dynamic Array IFC chips and the BioMark system (Fluidigm). Fluidigm real‐time PCR analysis software was used to calculate cycle threshold (C_t) values for each qPCR.

Tissue sections were thawed at room temperature and moved (with an autoclaved tweezer) to a 2 mL Eppendorf tube containing 400 μL lysis buffer (Beckman Coulter Lysis LBE, Cat no C39467) and one 5-mm Stainless steel bead (Qiagen, Cat no 69989). Next, 20 μL proteinase K was added (Beckman Coulter, Cat no C42096) and samples homogenized with an SPEX Sample Prep1600 MiniG, starting at 1,000 RPM for 4 min. Homogenized samples were next incubated for 30 min at 37°C and then placed at −80°C overnight to reduce foam. RNA was finally extracted using an automated protocol on a Biomek i7 (RNAdvance Cell v2, Cat no A47943).
RNA quality and concentration were measured on an Agilent Fragment Analyzer and the amount of input RNA used for cDNA synthesis normalized. cDNA and pre-amplification (12 cycles) was carried out according to the manufacturers recommendations (Fluidigm, Preamp and Reverse Transcription master mix, Cat no 100–6301) and gene expression levels determined using the Biomark HD system (Fluidigm, GE Dynamic Array Reagent Kit, Cat no 100–6267) with the following TaqMan probes (Thermo Fisher Scientific); Malat1 (Cat no Mm01227912_s1), Beta-actin, (Cat no Mm02619580_g1), Hprt1 (Cat no Mm03024075_m1), and Rplp0 (Mm00725448_s1).