title: GSM9523544: Mouse, ANDP Mutant 2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from the frontal cortex of WT and HET mice (n = 7), using the RNeasy Mini Kit (Qiagen; 74106) following the manufacturer's protocol. RNA concentration was determined with the Qubit RNA Broad Range Assay Kit (Invitrogen; Q10211). RT-PCR was used to confirm a selection of genes from the RNA sequencing experiment by converting 1 μg of total extracted RNA to cDNA using the SuperScript III Reverse Transcriptase kit (Invitrogen; 18080093). Primer efficiencies were optimized using a standard dilution curve method on pooled cDNA samples from wild-type and mutant mice (90% > E > 110%). RT-PCR was performed in triplicate using the CFX384 Touch Real-Time PCR Detection System (BioRad; 1855484) and the Takyon™ No ROX SYBR 2X MasterMix (Eurogentec; UF-NSMT-B0701) with primers listed in additional file S2. Reference gene stability was assessed using the geNorm method in qbase+ (Biogazelle), after which Actb, Gapdh and Rpl13a were selected for normalization. Library preparation was performed with the Novogene NGS RNA Library Prep Set (PT042). Using the sequencing platform Illumina Novaseq 6000 with sequencing strategy PE150. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.