Gene expression profile at single cell level of islets from two -cell specific knockout mouse models (ZKO-Mip and ZKO-Rip), littermate controls (Zzef1f/f), and the MIP-Cre mice fed with normal-chow diet (NCD).

Islet consists of multiple cell types that work in concert to orchestrate their endocrine function. To rule out of direct effect of diet or phenotype, we performed comparative single-cell RNA-sequencing (scRNA-seq) on 12-week ZKO-Mip, ZKO-Rip mice, littermate controls (Zzef1f/f) and MIP-Cre mice islets fed with normal-chow diet (NCD) by Chromium Next GEM Single Cell 3 Reagent Kits. Briefly, two -cell specific knockout mouse models (ZKO-Mip and ZKO-Rip) were generated by crossing the C57/BL6N mice harboring floxed exon 15 (Zzef1f/f) with MIP-Cre or RIP-Cre mice. Each islet sample was gathered from no less than three mice with the same genotype. Then, purified islets were treated with 2 ml 0.05% TryLE at 37 for 4 min, followed by gently pipetting for about 20 times. The cell suspension is stained with 0.4% trypan blue to assess cell viability under microscopic observation. The sorted cells with greater than 85% viability are qualified for 3 digital gene expression by profiling ~10,000 individual cells per sample using 10x Chromium Single-Cell 3 and V(D)J library construction (10x Genomics, USA), according to the manufacturers instructions.胰岛（Islet）由多种协同作用的细胞类型构成，共同调控其内分泌功能。为排除饮食或表型的直接影响，我们采用Chromium Next GEM单细胞3'试剂试剂盒（Chromium Next GEM Single Cell 3 Reagent Kits），对饲喂正常饲料（NCD）的12周龄ZKO-Mip、ZKO-Rip小鼠、同窝对照（Zzef1f/f）小鼠及MIP-Cre小鼠的胰岛开展了比较性单细胞RNA测序（scRNA-seq）。简言之，两种细胞特异性敲除小鼠模型（ZKO-Mip与ZKO-Rip）通过将携带两侧带有loxP位点的第15号外显子的C57/BL6N小鼠（Zzef1f/f）与MIP-Cre或RIP-Cre小鼠杂交构建所得。每份胰岛样本均取自至少3只相同基因型的小鼠。随后，将纯化后的胰岛置于2 mL 0.05% TryLE中，于37℃下消化4分钟，随后轻柔吹打约20次。将细胞悬液用0.4%台盼蓝染色，通过显微镜观察评估细胞活力。经筛选获得活力高于85%的细胞后，我们参照制造商的操作指南，使用10x Chromium单细胞3'及V(D)J文库构建试剂盒（10x Genomics，美国），对每份样本中的约10,000个单个细胞进行测序分析，以完成数字基因表达谱构建。