conditional inactivation of DYRK1A in the Dlx5 domain at embryonic forebrain at E15.5
we analyzed gene expression in E15.5 Dyrk1aDlxcre/+ forebrains and compared it to control littermates. Overall design: Forebrains from control (wt) and mutant (Dyrk1a<DlxCre/+>) mice (n=3 per genotypes) were microdissected at the age of 15.5 days post coitum (E15.5) from the Dyrk1a<DlxCre/+> line. Then RNA was extracted using Trizol and Qiagen RNeasy Minikit following the manufacturer protocol, and sent for single-end bulk RNA-Seq sequencing. The preparation of the libraries was done by the GenomEast platform, using the TruSeq Stranded Total RNA Sample Preparation Guide PN 15031048. Total RNA-Seq libraries were generated from 150-300 ng of total RNA using TruSeq Stranded Total RNA LT Sample Prep Kit with Ribo-Zero Gold (Illumina, San Diego, CA), according to manufacturer's instructions. Following purification, the depleted RNA was fragmented into small pieces using divalent cations at 94oC for 2 minutes. Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis. The molecule extracted from the biological material were polyA RNA. The sequencing was performed by the platform using Illumina Hiseq 4000.  The alignment was performed on the mouse GRCm38 release 93 using HISAT2 87 with the options -p 8 –dta, -t –summary-file –no-unal, --no-hd. Samtools version 1.9 (http://www.htslib.org/) was used to generate the bam file and index and the counts were generated using HTSeq-Count 0.9.1 88. Using RseQC 89 we checked that the percentage of mapped reads distributed in each genomic feature followed an optimal distribution (exon, 5'UTR exon, 3' UTR exon, Intron, Intergenic…). Then differential expression analysis was done using FCROS 90. Representation of protein-protein interaction network was obtained from mysregulated genes using Cytoscape 91.