Serum Reponse of CF1 primary MEFs , E13. Serum Reponse of CF1 primary MEFs , E13

The goal of this study was to find the longitudinal transcriptional response of Mouse Embryonic Fibroblast (MEF) primary cells during the progression of a cell cycle. Using RNASeq method (single-end 50-bp chemistry on Illumina Hi-seq 2500 instrument in high-throughput mode), we measured the transcriptional response for around two cell-cycles. Using the fold-change (with respect to average response before serum addition at t = 0) time-series data, we first identified, without using a priori knowledge, the duration and timing of cell cycle phases using a change-point detection algorithm. Next, using Least Absolute Shrinkage and Selection Operator (LASSO) and Estimation Stability with Cross Validation (ES-CV), we were able to, without any prior biological knowledge, extract information on the phase-specific causal interaction of cell cycle genes, as well as temporal interdependencies of biological mechanisms through a complete cell cycle. Overall design: MEF cells were serum starved for 36 hours (Serum Starved Conditions - 0.5% FCS, DMEM High Glucose, L-glutamine, Penicillin Streptomycin, fungizone, non-essential amino acids) prior to adding serum at t = 0 hr. At t = 0, FCS was added to 20% level by volume to stimulate cell growth. Cells were extracted using Trizol RNA isolation protocol with scraping. Total RNA was isolated using Zymo Research Direct-zol 96 RNA (R2054) kit. TruSeq Stranded mRNA HT Kit (RS-122-2101) was used for library preparation. mRNA was sequenced (single-end 50-bp) using Illumina HiSeq 2500 in high throughput mode, with 12 samples per lane. Data processing steps are: 1) Demultiplexing using Casava [configureBclToFastq.pl --mismatches 1], 2) Mapping/Alignment using RNA-STAR [STAR --genomeDir mm9 --sjdbGTFfile mouse_splice.sjdb --outSammode FULL --outFilterType BySJout], and 3) Normalization and Quantification using Homer [analyzeRepeats.pl rna mm9 -count exons -strand both].

本研究旨在探究小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblast, MEF）原代细胞在细胞周期进程中的纵向转录响应。我们采用RNA测序（RNASeq）技术（于Illumina HiSeq 2500仪器上以高通量模式运行的单端50bp测序方案），对约两个完整细胞周期的转录响应进行了检测。基于以t=0时刻血清添加前的平均转录响应为参照的倍数变化时序数据，我们首先通过变点检测算法，在不依赖任何先验知识的前提下，确定了细胞周期各时相的持续时长与发生时序。随后，借助最小绝对收缩和选择算子（Least Absolute Shrinkage and Selection Operator, LASSO）以及交叉验证估计稳定性（Estimation Stability with Cross Validation, ES-CV），我们无需借助任何先验生物学知识，即可提取得到细胞周期基因的时相特异性因果互作信息，以及完整细胞周期内生物学机制的时间级联依赖关系。总体实验设计如下：实验前，将MEF细胞置于血清饥饿培养条件（含0.5%胎牛血清的高糖DMEM培养基，添加L-谷氨酰胺、青霉素-链霉素、两性霉素B及非必需氨基酸）中培养36小时，于t=0小时添加终体积分数20%的胎牛血清以刺激细胞增殖。采用Trizol试剂结合细胞刮取法提取细胞总RNA，并使用Zymo Research Direct-zol 96 RNA（R2054）试剂盒完成总RNA纯化。使用TruSeq Stranded mRNA HT Kit（RS-122-2101）构建测序文库。以单端50bp模式在Illumina HiSeq 2500仪器上开展高通量测序，每泳道可容纳12个样本。数据处理步骤如下：1. 使用Casava软件进行双端序列拆分解码，命令为configureBclToFastq.pl --mismatches 1；2. 使用RNA-STAR软件进行序列比对，命令为STAR --genomeDir mm9 --sjdbGTFfile mouse_splice.sjdb --outSammode FULL --outFilterType BySJout；3. 使用Homer软件进行标准化与定量分析，命令为analyzeRepeats.pl rna mm9 -count exons -strand both。