title: Illumina NovaSeq 6000 paired end sequencing; Transcriptomic profile of porcine corpus luteum after PPAR gamma ligands treatment
design: Transcriptomic profile of porcine corpus luteum after PPAR gamma ligands treatment
library_name: Sample 19_p
library_construction_protocol: In the laboratory, the CL was separated from the ovary, purified and placed on ice in a sterile petri dish. The CLs were cut into small pieces (100 ± 10 mg, collected in duplicate from each experimental replicate) and washed twice with sterile PBS. Each tissue explant was placed in a sterile culture vial with 2 mL of medium M199 (Sigma Aldrich, St. Louis, MO, USA), supplemented with 0.1% BSA fraction V (Roth, Germany) with antibiotics: nystatin (120 IU/mL; Sigma Aldrich) and gentamicin (40 mg/mL; Sigma Aldrich). The explants were pre-incubated for 2 h in a water bath at 37°C in an atmosphere 95% O2 and 5% CO2 and then treated for 6 h with PPARγ ligands: pioglitazone (P; synthetic agonist; 10 µM; Cayman Chemical Company) and T0070907 (T; antagonist; 10 µM, Cayman Chemical Company). The PPARγ ligands concentration and incubation times were selected according to our preliminary study and previous reports as described previously [12,14,15]. The controls (without the treatments) with culture medium and DMSO (solvent for PPARγ ligands) were also included. After the incubation, the tissue explants were washed with PBS and frozen at -80°C until RNA-Seq and quantitative Real‐Time PCR analysis. Total RNA from 24 samples (4 pigs x 2 stages of the estrous cycle x 3 treatments) was isolated using the “RNeasy Mini Kit” (Qiagen, Germany) in accordance to the manufacturer's protocol. The purity and concentration of the isolated RNA was determined with the Tecan Infinite M200 plate reader (Tecan Group Ltd., Switzerland). Sample degradation was evaluated in the Agilent Bioanalyzer 2100 (Agilent Technology, USA). The samples with RNA Integrity Number (RIN) >7 were selected for the next analyses. Poly(A) RNA sequencing library was prepared following Illumina's TruSeq-stranded-mRNA sample preparation protocol. Poly(A) tail-containing mRNAs were purified using oligo-(dT) magnetic beads with two rounds of purification. Subsequently, poly(A) RNA was fragmented using a divalent cation buffer in elevated temperature and then  poly(dT) oligonucleotides were applied to transcribe RNA into cDNA. Next, the cDNA was processed to 3' tail adenylation and adapter ligation. The reverse transcription during the library preparation was strand-specific. Finally, pooled libraries were followed by sequencing procedure. Quality control analysis and quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip.