Transcriptome of murine lumbar spinal cord at E12.5 in control and DOT1L cKO through Wnt1cre2 (B6.Cg-E2f1Tg(Wnt1-cre)2Sor/J, Jackson Laboratory #022501)

DOT1L has a proven function in the cortical and cerebellar development of the mouse model, but has never been studied in the developing spinal cord. Here, we created a transgenic mouse line for Dot1l conditional knock-out in the spinal cord over neurogenesis. We investigated the changes in the trascriptome by performing bulk RNAseq of lumbar spinal cords in controls and Dot1l-cKO. The DEG analysis of cKO littermates revealed a higher degree of differentiation profile as opposed to the controls, concurrent with decreased cell proliferation and altered axonal projection and interneuron migration, supporting the importance of DOT1L activity in the developing spinal cord. 10 mouse embryos (5 controls and 5 Dot1l-cKOs) from 2 litters at E12.5 (3 n per condition from the first, 2 n per condition from the second). Lumbar spinal cord isolation is followed by RNA extraction of single samples, library preparation and sequencing.

DOT1L已被证实可在小鼠模型的大脑皮层与小脑发育过程中发挥功能，但此前尚未在发育中的脊髓中开展相关研究。本研究构建了可在脊髓神经发生阶段对Dot1l进行条件性敲除（conditional knock-out，cKO）的转基因小鼠品系。我们通过对对照组与Dot1l-cKO组小鼠的腰段脊髓进行批量RNA测序（bulk RNAseq），探究了两组样本的转录组（transcriptome）变化。对同窝cKO小鼠的差异表达基因（differentially expressed genes，DEG）分析结果显示，与对照组相比，cKO组小鼠呈现出更为显著的分化特征，同时伴随细胞增殖水平降低、轴突投射异常以及中间神经元迁移改变，这进一步证实了DOT1L活性在发育中脊髓内的重要作用。本实验共使用来自2窝的10枚小鼠胚胎，其中对照组与Dot1l-cKO组各5枚，胚胎阶段为胚胎发育第12.5天（E12.5）：第一窝每个实验组与对照组各3枚样本，第二窝每个实验组与对照组各2枚样本。实验流程为：先分离腰段脊髓，随后对单个样本进行RNA提取、文库制备及测序。