title: Illumina NovaSeq 6000 paired end sequencing; The impact of chemerin on transcriptomic profile of swine luteal cells during mid-luteal phase of estrous cycle
design: The impact of chemerin on transcriptomic profile of swine luteal cells during mid-luteal phase of estrous cycle
library_construction_protocol: Gilts were obtained from a private breeding farm, and during the mid-luteal phase of the estrous cycle (days 10–12 of the cycle) were used for the research. Gilts were daily observed for estrus behavior in the presence of a boar. The day when the symptoms of the second estrus appeared was designated as day 0 of the estrous cycle. Additionally, ovarian morphology has been verified to confirm the phase of the cycle (PMID: 5692889). Immediately after humanitarian slaughter, ovaries were collected and transported to the laboratory in ice-cold PBS supplemented with 100 IU/mL penicillin and 100 µg/mL streptomycin. Luteal cells were isolated from the slaughter material. Corpora lutea were dissected from the ovaries, divided into smaller fragments, and dispersed in Ham's F-12 nutrient mixture (Sigma-Aldrich, St. Louis, MO, USA) containing 1% of bovine serum albumin fraction V (Sigma-Aldrich, St. Louis, MO, USA) and mix of antibiotics (Sigma-Aldrich, St. Louis, MO, USA). Swine corpora lutea were enzymatically decomposed 4–6 times using 0.125% trypsin (Sigma-Aldrich, St. Louis, MO, USA) solution at 38 °C, centrifuged for 10 min at 300 × g at 21 °C, and washed three times. Luteal cells were filtered from trypsinized tissue residues with a 75 µm mesh nylon filter and resuspended in fresh Ham's F-12 mixture. The number of cells was estimated with a hemocytometer and their viability of ≈90% was determined with 0.4% trypan blue dye (Sigma-Aldrich, St. Louis, MO, USA) exclusion. Luteal cells were resuspended at the concentration of 2 × 10^6/2 mL in Ham's F-12 medium supplemented with 20% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 1% bovine serum albumin fraction V, and antibiotics. The cells were pre-incubated in a humidified incubator with 95% air and 5% CO2 atmosphere for 48 h. The serum-containing culture medium was rejected, and luteal cells were flushed with serum-free Ham's F-12 nutrient mixture. Porcine luteal cells were cultured for 24 h in F-12 mixture with 1% bovine serum albumin fraction V, antibiotics, and with or without the recombinant human chemerin (cat. no. 268-10032; RayBiotech, Peachtree Corners, GA, USA) at the concentration of 200 ng/mL for the chemerin-treated and control group (n = 5), respectively. From in vitro cultures of luteal cells treated and untreated with chemerin, total RNA was extracted with the use of RNeasy Mini Kit (Qiagen, Germantown, MD, USA) with DNase included in RNase-free DNase Set (Qiagen, Germantown, MD, USA), in accordance to manufacturer's procedure. The purity (A260/A280) and quantity (wavelength 260 nm, A260) of the obtained RNA was estimated spectrophotometrically with an Infinite M200 Pro (Tecan, Männedorf, Switzerland). RNA integrity was tested using the Bioanalyzer 2100 (Agilent Technology, St. Clara, CA, USA). RNA integrity number in a range between 8 and 10 qualified isolated RNA for the transcriptome high-throughput sequencing (RNA-Seq) library preparation. The strand-specific sequencing libraries were prepared separately from each sample RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA), ac-cording to Shen et al. (doi:10.3390/genes7070034) recommendations.