RNA-sequencing experiments comparing P0 control and RXRG/RARb double mutants frontal cortex

To understand the functional significance of RA signaling through the RXRG/RARB heterodimer in the developing cortex, we performed RNA-seq experiments comparing different regions of the P0 mouse frontal cortex (mPFC, secondary motor cortex (MOs)/ primary motor cortex (MOp), and OFC ) in Rarb and Rxrg dKO and WT littermate controls. Overall design: Freshly dissected P0 brains were frozen at -80C, and moved to RNALater 12-16 hrs prior to further dissection. Medial frontal, motor, and orbitofrontal regions were microdissected. RNA was isolated using Rneasy plus micro kit (Qiagen), and cDNA libraries were created using SMART-seq v4 Ultra Low Input Kit (Takara) and Nextera XT DNA library Prep Kit (Illumina). Libraries were normalized and sequenced at the Yale Center for Genomic Analysis (YGCA) using the NovaSeq with 100 bp paired end reads.

为阐明由RXRG/RARB异二聚体（RXRG/RARB heterodimer）介导的视黄酸（retinoic acid, RA）信号通路在发育大脑皮层中的功能意义，本研究开展了RNA测序（RNA-seq）实验，对比了P0小鼠前额叶皮层（medial prefrontal cortex, mPFC）、次级运动皮层（secondary motor cortex, MOs）/初级运动皮层（primary motor cortex, MOp）以及眶额皮层（orbitofrontal cortex, OFC）不同区域在Rarb与Rxrg双基因敲除（double knockout, dKO）小鼠及其野生型（wild type, WT）同窝对照中的转录组特征差异。实验整体设计如下：新鲜解剖获取的P0小鼠脑组织先置于-80℃冷冻保存，后续解剖前需在RNALater中放置12至16小时。通过显微解剖技术分离内侧前额叶、运动皮层及眶额皮层目标区域。采用RNeasy Plus Micro试剂盒（Qiagen）提取总RNA，使用SMART-seq v4超低起始量Input试剂盒（Takara）与Nextera XT DNA文库制备试剂盒（Illumina）构建cDNA文库。文库经标准化处理后，由耶鲁大学基因组分析中心（YGCA）依托NovaSeq测序平台进行100 bp双端测序。