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. 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].