Next-generation sequencing analysis of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts (MEFs). Next-generation sequencing analysis of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts (MEFs)

Purpose: The goals of this study are to use NGS to perform transcriptome profiling (RNA-seq) to find the difference of the transcriptomes of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts. Methods: MEFs were reprogramed into neurons by small chemical cocktails. Small chemical molecules were dissolved and diluted in DMSO and used at the following final concentrations: ISX9: 20 mM; Forskolin: 50 mM; CHIR99021: 20 mM; and I-BET151: 2 mM. In addition to the small molecules, the neuron induction medium (Neurobasal Medium) contains the following supplements: N2 (1X) and B27 (2X) supplements, GlutaMAX (1X), penicillin-streptomycin (100 µg/ml), bFGF (20 ng/ml), 100 µM cAMP, Non-essential Amino Acid (1X) and Trace element B (1X). MEFs were seeded to Matrigel-coated plate (1:30 dilution in pre-cold PBS and coat overnight at 4 oC, at a density of 200,000 cells per well in 6-well plate and 10,000 cells/well in 96 well plate. The MEFs were cultured in DMEM until confluent. When the cells are confluent, the DMEM was replaced with neuron induction medium with 4 small molecules. The induction medium was refreshed every two days for the first week and every 3 days for the remaining induction period until day 20. 3 biological replicates of total RNA were used for RNA-seq library preparation. Results: Using an optimized data analysis workflow, we identified neuronal gene programs induced during direct reprograming. The induction of neuronal programs were impaired in neurons from Actb-/- mouse embryonic fibroblasts. Conclusions: Our study shows that neuronal program induction was impaired in chemically induced neurons from Actb-/- mouse embryonic fibroblasts. Overall design: mRNA profiles of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts were generated by deep sequencing, in 3 biological replicates.

### 研究目的
本研究旨在利用下一代测序（Next-Generation Sequencing，NGS）开展转录组分析（transcriptome profiling，RNA-seq），对比野生型（wild type，WT）、Actb基因杂合敲除（Actb+/-，HET）及Actb基因纯合敲除（Actb-/-，KO）小鼠胚胎成纤维细胞经化学诱导获得的神经元的转录组差异。

### 实验方法
本研究通过小分子化合物组合将小鼠胚胎成纤维细胞（mouse embryonic fibroblasts，MEFs）重编程为神经元。将小分子化合物溶解于二甲基亚砜（DMSO）中并稀释，终浓度设置如下：ISX9：20 mM；Forskolin：50 mM；CHIR99021：20 mM；I-BET151：2 mM。除上述小分子外，神经元诱导培养基（Neurobasal Medium）还添加以下组分：N2（1×）、B27（2×）添加剂，GlutaMAX（1×）、青霉素-链霉素（100 µg/ml）、碱性成纤维细胞生长因子（basic fibroblast growth factor，bFGF，20 ng/ml）、100 µM 环磷酸腺苷（cyclic adenosine monophosphate，cAMP）、非必需氨基酸（1×）及微量元素B（1×）。
将MEFs接种于基质胶（Matrigel，用预冷磷酸盐缓冲液（PBS）按1:30比例稀释，4℃包被过夜）包被的培养板中：6孔板每孔接种200,000个细胞，96孔板每孔接种10,000个细胞。将MEFs置于DMEM培养基中培养至细胞汇合，待细胞汇合后，将DMEM替换为含4种小分子的神经元诱导培养基。诱导期间，前7天每2天更换一次培养基，后续诱导阶段每3天更换一次，直至第20天。收集3份生物学重复的总RNA用于构建RNA-seq文库。

### 实验结果
通过优化的数据分析流程，我们鉴定出直接重编程过程中被诱导激活的神经元基因表达程序。在Actb基因纯合敲除小鼠胚胎成纤维细胞来源的化学诱导神经元中，神经元基因程序的激活受到损伤。

### 研究结论
本研究证实，Actb基因纯合敲除小鼠胚胎成纤维细胞经化学诱导获得的神经元中，神经元基因程序的激活存在缺陷。

### 整体实验设计
本研究通过深度测序（deep sequencing）生成了野生型、Actb基因杂合敲除及纯合敲除小鼠胚胎成纤维细胞经化学诱导得到的神经元的mRNA表达谱，每组设置3份生物学重复。