Total RNA was isolated from brains of E15 embryos using TRIzol (Invitrogen) protocol, followed by an analysis of quality with Bioanalyzer (Agilent) and quantity Qubit (Invitrogen). Subsequently, those samples were submitted to library construction using TruSeq Stranded mRNA HT Sample Prep Kit (Illumina), in which mRNA is enriched across magnetic beads with oligo(dT). This enriched mRNA template is converted to double-stranded cDNA, followed by AMPure XP bead purification, end repair, and A-tail addition. For each sample, an index was associated, and so, the library was amplificated by PCR to enable cluster generation for sequencing at HiSeq 2500 (Illumina). The RNA-seq data were mapped to the Genome Reference Consortium Mouse Build 38 (GRCm38–mm10) with STAR 2.5.4b algorithm (44) after count tables for each gene was generated by HTSeq. The differential expression of genes was done in edgeR package. Gene Ontology enrichment analysis was performed with the R package GAGE 2.3. From the list of differentially expressed genes (P < 0.05 and log fold change, ≤−1 or ≥1), we performed gene set enrichment analysis using ensemble of gene set enrichment analyses and derived a protein-protein interaction network with NetworkAnalyst.

A list of differentially expressed genes is available in table S5. For validation, five differentially expressed genes were selected from Fig. 4 (C and D) and analyzed by RT-qPCR (Ccr1, Shank1, Galr1, Grin1, and Slc6a7). For this purpose, purified RNA samples were submitted to retrotranscription followed by qPCR reaction in a one-step reaction protocol. The constitutive gene used was GAPDH, which showed good performance in mice in E15 (45). Results of relative expression were expressed by 2ΔCT. We used the GoTaq 1-Step RT-qPCR System (Promega) for Ccr1 and Shank1, and for quantification of Galr1, Grin1, and Slc6a7, the LightCycler 480 System (Roche Life Sciences) was used. Results are presented in fig. S15.

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