| Overall design |
Seven WT- and 5 Mtor-CKO ovarian samples were collected from 6-month old mice that were initially primed with eCG (5 IU/mouse) for 46 h. Total RNA was extracted with RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to manufacturer's instructions, and 500 ng was then taken out from each of them and subjected to mRNA isolation using NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, Ipswich, MA, USA). The mRNA library was constructed using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA) according to the instruction manual, which includes sequential RNA fragmentation, reverse transcription using random primers, second strand cDNA synthesis, end repair, dA-tailing, adapter ligation, U excision, and PCR enrichment. The ovarian mRNA libraries were sequenced on an Illumina HiSeq X Ten platform with 150bp pair-end reads. All reads passed filter were trimmed to remove low-quality bases and adaptor sequences. Reads were then aligned to the mm10 reference genome using tophat2 (v2.0.13), and FPKMs were calculated and normalized using cufflinks (v2.2.1). The differentially expressed genes were calculated using default parameter of cuffdiff (v2.2.1). Hierarchical clustering was carried on log2(FPKM+1) across samples. Genes used for clustering were selected by maximum FPKM≥1 and with top 10% standard deviation of log2(FPKM+1).
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