RNA-Seq analysis of mouse dopamine neurons identifies transcriptional repression of PlexinC1 by Lmx1a and Lmx1b

In order to identify axon guidance molecules regulated by Lmx1a and Lmx1b RNA sequencing was performed on three biological replicates for the control and four biological replicates for Lmx1a/b cKO mice. For each sample, two technical replicates were performed. For each E15.5 embryo used, VTA and SNpc were dissected from 8 antero- posterior levels across the entire dopaminergic domain as revealed by TH immunostaining. Following RNA extraction, RNAseq libraries were constructed using the Illumina TruSeq Stranded RNA protocol with oligo dT pulldown and sequenced on Illumina HiSeq2500 by 150-bp paired-end sequencing. One sample was excluded before the sequencing because the amount of RNA was too low to measure the RNA quality.为鉴定受Lmx1a与Lmx1b调控的轴突导向分子，本研究为对照组小鼠设置3次生物学重复样本、为Lmx1a/b条件性敲除（conditional knockout, cKO）小鼠设置4次生物学重复样本，并开展RNA测序实验。每个样本均设置2次技术重复。针对每一枚胚胎发育第15.5天（E15.5）的胚胎，研究人员通过酪氨酸羟化酶（Tyrosine Hydroxylase, TH）免疫染色标记全脑多巴胺能区域，随后从该区域的8个前后轴水平位置中分离腹侧被盖区（Ventral Tegmental Area, VTA）与黑质致密部（Substantia Nigra Pars Compacta, SNpc）。RNA提取完成后，采用搭载寡聚dT富集步骤的Illumina TruSeq链特异性RNA建库试剂盒构建RNA测序（RNA sequencing, RNA-seq）文库，并在Illumina HiSeq2500测序平台上开展150 bp双端测序。测序前有1个样本因RNA总量过低无法检测RNA质量而被排除。