Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA was released from small number of oocytes upon incubation in water with RNase inhibitor for 5 min at 85°C. RNA was reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Fermentas). Real-time PCR was carried out on LightCycler 480 System (Roche). Each reaction (10 µl) consisted of 5 µl Maxima® SYBR Green qPCR Master Mix (Fermentas), 0.4 µl mixed gene-specific forward and reverse primers (30 µM each) and 2 µl diluted cDNA. Each sample was run in triplicate. Total RNA was extracted from 25 oocyte using PicoPure RNA Isolation Kit with on-column genomic DNA digestion according to the manufacturer’s instruction (Thermo Fisher Scientific, Philadelphia, PA). Each sample was spiked in with 0.2pg synthesized Renilla Luciferase mRNA before extraction as normalization control. RNAseq libraries were constructed by using Ovation RNA-seq system V2(NuGEN, San Carlos, CA) followed by Ovation Ultralow Library system(DR Multiplex System, NuGEN, San Carlos, CA).