Quant it ribogreen rna assay kit

Gene expression profiling was previously described in detail in an earlier work.33 In brief, RNA was initially purified using Qiagen PaxGene Blood RNA MDx Kits in PAXgene tubes containing whole blood. The Illumina TotalPrep RNA amplification kit (Life Technologies) was then used to biotinylate the RNA. Afterward, quantity and quality of the RNA were assessed using the Quant‐iT RiboGreen RNA Assay Kit and Bioanalyzer RNA Nano chip assay (Agilent, Santa Clara, CA), respectively. Expression data from the Human HT‐12v3 Expression BeadChip (Illumina) were used for assessment of the genes of interest. QC was carried out using the Illumina Genome Studio software, and probes detected in >50% of samples and that had a detection P<0.05 were utilized. Plate, nested chip, and sample effects were also assessed using variance components and principal components analyses, and outliers at each level were excluded. Robust multichip average methods were used to log₂ transform and quantile normalize the expression data.

Total RNA was isolated from FAC‐sorted GFP‐positive cells from Hes5::GFP and Tbr2::GFP transgenic lines using TRIzol reagent. A time course was performed with NSCs, BPs, and NBNs isolated at each time point during development from E10.5 to postnatal day 1 (PN) or as specified in the Fig 1A. Samples were analyzed for their integrity and concentration using Agilent 2100 Bioanalyzer and Quant‐IT RiboGreen RNA Assay Kit. Sequencing libraries were prepared with the Illumina TruSeq RNA Library Prep Kit v2 according to Illumina's instructions. After quality control (Fragment Analyzer, AATI), libraries were pooled and loaded on an Illumina flow cell for cluster generation (HiSeq SR Cluster Kit v4 cBot). Libraries were sequenced SR50 on the HiSeq 2500 system (HiSeq SBS Kit V4) following the manufacturer's protocols.

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.