Bovine v2 gene expression microarrays 4 44 k

We selected a panel of nine candidate genes (CCL2, HCK, F11R, CD8A, PDIA3, LGMN, HSPA1A, IL18, NFKBIA), which in earlier analyses using expression microarrays (Bovine (V2) Gene Expression Microarrays 4 × 44K, Agilent, USA) revealed differences in expression level in non-infected mammary gland secretory tissue vs. those infected with coagulase-positive or coagulase-negative staphylococci (Kościuczuk et al., 2017) . The potential gene promoter regions were selected using MatInspector software (Genomatix). The methylation level of CpG sites in selected genes was determined by bisulfite sequencing PCR (BSP), with primers designed with Methyl Primer Express ® Software v1.0 (Applied Biosystems Software). The amplification reaction was performed in 20 μl volumes, using Hot Start Taq ® polymerase (Qiagen). A two-round BSP amplification protocol was used. The thermal amplification conditions and the primer sequences are presented in Table 1. After amplification, BSP products were exposed to exonuclease I and alkaline phosphatase (Thermo Fisher Scientific), and sequenced using a BigDye ® Terminator v3.1 kit (Thermo Fisher Scientific), according to the standard protocol. The sequencing products were electrophoresed on a 24-capillary 3130xl genetic analyzer (Applied Biosystems).