RNA sequencing on Postnatal day (P) 7 wildtype and Zmiz1-Knockout cortex

Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 mutations association with neurodevelopmental disorders such as ASD, ADHD, and intellectual disability, its role in physiological and pathological neurodevelopment is significantly unknown. Here, we use murine model to knockout Zmiz1 using Emx1Cre and performed RNA sequencing on P7 cortex to profile transcriptional changes upon Zmiz1 deletion. Timed pregnancy was carried out. Mice was euthanized using CO2 and embryos brain were extracted at E15.5. Cortex was dissected and dissociated into single cell suspension using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, 130-092-628) following manufacture instruction. Intermediate progenitors were magnetically selected and isolated using Anti-Prominin-1 MicroBeads, mouse (Miltenyi Biotec, 130-092-333). Total RNA was extracted from wildtype and Zmiz1-KO samples. RNA concentration and RNA integrity number were determined. RNA library was prepared, quantified, and verified using TruSeq RNA Library Prep Kit v2, Qubit dsDNA High Sensitivity Assay kit and Bioanalyzer DNA1000 assay kit respectively. Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 on a Nextseq1000/2000. RNA-seq data analysis was performed using illumina BaseSpace Sequence Hub. Briefly, sequenced reads were aligned to mouse (mm10) reference genome with RNA-Seq alignment tool (STAR aligner) and differentially expressed genes (DEG) were determined using the RNA-Seq Differential Expression tool (version 1.0.1). Results: We found 114 differentially expressed genes of which 35 genes were upregulated while 69 genes were downregulated. Downregulated genes were enriched in biological processes such as forebrain deveopment, axon development, neuron differentiation etc. Conclusions: We assessed P7 cortex specific transcriptional profile changes which are potentially regulated by Zmiz1. P7 WT and Zmiz1-KO cortex RNA sequencing研究目的：含锌指MIZ型结构域1（Zinc Finger MIZ-Type Containing 1, Zmiz1）属于PIAS家族蛋白，可作为Notch、雄激素受体（Androgen Receptor, AR）、p53、雌激素受体（Estrogen Receptor, ER）以及Smad3/4的转录共激活因子发挥功能。尽管Zmiz1突变与自闭症谱系障碍（ASD）、注意缺陷多动障碍（ADHD）及智力障碍等神经发育障碍相关，但其在生理及病理神经发育过程中的作用仍有待阐明。本研究利用Emx1Cre介导的小鼠模型敲除Zmiz1，并对出生后第7天（P7）的小鼠皮层进行RNA测序，以解析Zmiz1缺失后的转录组变化。 实验采用定时孕鼠模型：通过CO₂处死小鼠，于胚胎期15.5（E15.5）提取胚胎脑组织。按照操作说明书，使用神经组织解离试剂盒（P）（Miltenyi Biotec, 130-092-628）将皮层解剖并解离为单细胞悬液；通过小鼠抗Prominin-1磁珠（Miltenyi Biotec, 130-092-333）磁选分离中间祖细胞。从野生型（WT）及Zmiz1敲除（Zmiz1-KO）样本中提取总RNA，检测RNA浓度与RNA完整性数。分别使用TruSeq RNA文库制备试剂盒v2、Qubit dsDNA高灵敏度检测试剂盒与生物分析仪DNA1000检测试剂盒完成RNA文库的构建、定量与验证。验证合格的样本使用NextSeq1000/2000测序仪，搭配NextSeq1000/2000 P2试剂（200循环）v3进行测序。 RNA测序数据分析依托Illumina BaseSpace序列分析平台完成：简要而言，将测序reads比对至小鼠参考基因组mm10，使用RNA-seq比对工具（STAR aligner）进行比对，并通过RNA-seq差异表达分析工具（版本1.0.1）鉴定差异表达基因（differentially expressed genes, DEG）。 研究结果：本研究共鉴定得到114个差异表达基因，其中35个基因上调，69个基因下调。下调基因富集于前脑发育、轴突发育、神经元分化等生物学过程。 研究结论：本研究解析了P7皮层中受Zmiz1潜在调控的特异性转录组变化。P7 WT与Zmiz1-KO皮层RNA测序