Gene expression profile of bovine endometrial cells under heat stress
Heat stress provide various effects on cellular functions. The aim of the present project is to know how the heat stress influence the gene expression profiles of bovine endometrial stromal and epithelial cells in vitro. Both types of cells were harvested from endometrium of uteri derived from slaughterhouse. After enzymatic digestion of the tissue, epithelial and stromal cells were isolated. These cells were cultured under heat stress condition (40.5ÂºC) or control (38.5 ÂºC). Total RNA extraction was conducted using the RNeasy Mini kit (74134, Qiagen, Redwood City, CA, USA), according to the manufacturer's instructions. The optical density A260/280 of RNA was measured by NanoDrop LITE (Thermo Fisher Scientific) and its value was approximately 2. The RNA integrity number (RIN) was assessed by Agilent 2100 Bioanalyzer (Agilent Thechnologies) and its value was above 8. To produce the cDNA libraries, 500 ng total RNA was used for mRNA isolation using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), then the library quality and quantity were determined using the Agilent 2100 Bioanalyzer and the Kapa Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA), respectively. Each cDNA library was sequenced on the NextSeq 500 (Illumina) using a High Output single end 1 Ã? 75 bp run (NextSeq 500/550 High Output Kit v2.5, Illumina) and 30 million raw reads per sample were generated. After base calling by RTA version 2.4.11, Fastq file was generated using bcl2fastq version 2.18.0.12 (Illumina), according to the manufacturer's instructions. Sequence data was filtered to discard adapter sequences, ambiguous nucleotides, and low-quality sequences. To count sequence reads, the remaining sequence data was aligned to the Bos Taurus genome sequence (ARS-UCD1.2/bosTau9). The expression values for each gene and statistical analysis of differentially expressed genes were determined using mapped sequence data. Filtering, mapping, and subsequent analysis were performed using the CLC Genomics Workbench software (Qiagen, Redwood City, CA, USA). Statistical significance was determined byempirical analysis using DGE tool. Differentially expressed genes (P < 0.05 or Fold Change >1.2) were used for further analyses.