The effect of acetic acid treatment to the cumulus cells during the porcine in vitro maturation.
Acetic acid is major substate founded at 1mM in porcine blood and FFs. This data is the RNA-seq of the cumulus cells cultured from 44h of in vitro maturation (IVM) treated with 1mM of acetic acid. The medium used for the in vitro maturation (IVM) of oocytes was Porcine oocyte medium (POM) (K Yoshioka et al. 2008; PMID: 18408352) supplemented with 10% v/v porcine follicular fluids (FF),0.5mM L-cysteine,10ng/ml epidermal growth factor (Sigma-Aldrich, St Louis, MO, USA) and 3mg/ml Polyvinyl-alcohol. Porcine FF was aspirated from the antrum follicles (3-5 mm in diameter) of gilts,  centrifuged (10,000 Ã? g for 5 min) and stored at -20Â°C until use. The cumulus cells oocyte complexes (COCs) were cultured in IVM medium containing 0 (Control),0.1 or 1mM of Acetic acid (10 COCs each 100ul drops) for 44h; the first half of 24h, the COCs were cultured with IVM medium containing 1mM of dbcAMP, 10 IU/ml equine chorionic gonadotropin (ASKA Pharma Co., Ltd., Tokyo, Japan), and 10 IU/ml human chorionic gonadotropin (Fuji Pharma Co., Ltd., Tokyo, Japan), and in the end half of 20h, COCs were cultured without hormone and dbcAMP. The concentration of acetic acid was set considering a previous report where FF contained acetic acid at 1mM. (R G Gosden et al. 1990; PMID: 2171975) The cumulus cells were separated from COCs. The total RNA extraction was conducted using RNAqueous-Micro (Thermo Fisher). Three samples were created for each experimental group using differential ovary series. The quality and concentration of total RNA were determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA quality index was 9.23. cDNA libraries of the RNA were prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina New England Biolabs). Concentration of the cDNA libraries were determined using agilent high sensitivity DNA kit and Bioanalyzer 2100 (Agilent Technology). The concentration of the cDNA libraries was reassessed using Kapa library quantification kit (Kapa Biosystem). The multiplexed sample was sequenced in single-read 75bp reads on the NextSeq 500 system (Illumina) with NextSeq 500/550 High Output Kit v2.5(Illumina). Image analysis, base calling, and quality filtering were performed using RTA version 2.4.11 (Illumina). Raw data were generated using bcl2fastq2-v2.20.0.422. (Illumina), according to the manufacturer's instructions.