Col2a1-Cre;Idh1LSL/+ embryonic growth plate - Bulk RNA-sequencing

As another method to determine differences between Idh1 mutant and control growth plates, we used Bulk RNA sequencing. Three E18.5 growth plates from our Idh1 mutant mice and three control mice were studied. Differential gene expression analysis revealed genes that were expressed in the distinct clusters, further supporting our single cell RNA data. In order to assess the Idh1 mutant expressing chondrocytes, we utilized Collagen2a1-Cre males crossed with Idh1 LSL/+ females. Growth plate chondrocytes from E18.5 distal part of femur and proximal part of tibia were collected and snap frozen. Total RNA was isolated and used for RNA-seq analysis. After sequencing and downstream analysis using Seurat, we observed distinct clusters of cells with Idh1 mutation Total RNA was isolated using Single Cell RNA Purification Kit and submitted to core facitly for library generation and sequencing为明确Idh1突变型与对照生长板之间的差异，我们采用批量RNA测序（Bulk RNA sequencing）技术。本研究选取3株Idh1突变型小鼠及3株对照小鼠的胚胎18.5天（E18.5）生长板样本开展分析。差异基因表达分析筛选出了在独特细胞簇中表达的基因，进一步验证了我们的单细胞RNA测序数据。 为评估表达Idh1突变的软骨细胞，我们将胶原2a1-Cre（Collagen2a1-Cre）雄性小鼠与Idh1 LSL/+雌性小鼠进行杂交繁育。收集胚胎18.5天小鼠股骨远端及胫骨近端的生长板软骨细胞，并进行快速冷冻处理。提取总RNA用于RNA测序分析。 完成测序及基于Seurat的下游分析后，我们观察到携带Idh1突变的细胞形成了独特的细胞簇。本次实验采用单细胞RNA纯化试剂盒（Single Cell RNA Purification Kit）提取总RNA，并将样本提交至核心实验平台进行文库构建与测序。