Bio imaging system

Total genomic DNA of the isolate was extracted according to the method of Wu et al. (2009 (link)). 18S rRNA gene fragment was amplified by PCR using the set of primers NS1 (5-GTAGTCATATGCTTGTCTC-3) and NS4 (5-CTTCCGTCAATTCCTTTAAG-3) designed to anneal to conserved regions of the fungi’s 18S rRNA. A 15-μL reaction volume included 1 μL (50 ng/μL) template, 7.5 μL 2× Taq PCR MasterMix, 5.9 μL ddH2O, and 0.3 μL of each primer (10 μmol/μL), performed in a Peltier Thermal cycler (M. J. Research, Inc, Japan) according to the following conditions: 35 cycles of 94 °C for 30 s (de-naturation), primer annealing at 55 °C for 50 s, and 72 °C for 150 s, and a final extension at 72 °C for 10 min. PCR products were then electrophoresed on 1.5% agarose gels using 1× TAE buffer (40 mM Tris acetate; 1 mM EDTA; pH 8.0), containing 200 ng/mL ethidium bromide to detect the product. Images were captured on a Syngene Bioimaging system (Syngene, U.K.). Sequencing of PCR products were conducted by excising the bands from the agarose and purifying the complementary DNA (cDNA) using AuPrep GelX kit (Life Technologies Ltd.). The purified cDNA was sequenced. The resulting sequences were analyzed and aligned using Blast Local Alignment Tool (BLAST) program at the National Centre for Biotechnology Information (NCBI).

ChIP assays were carried out using a Merck-ChIP Kit (Catalog number #17–371, Millipore, Merck, Germany) according to the manufacturer’s protocol. Briefly, cells were grown to 90% confluence in three 10 cm culture dishes and crosslinked with 1% formaldehyde at room temperature for 10 min. Glycine was used to quench excess formaldehyde, and the cells were scraped and pelleted. Chromatin was sonicated in lysis buffer on ice to 200–1000 bp using a sonicator (Sonics, USA) at 55–75% intensity four times. The sheared DNA lengths were confirmed via agarose gel electrophoresis. After sonication, the chromatin was incubated with an anti-SP1 antibody (Catalog number ab231778, ChIP Grade, Abcam) or IgG and rotated at 4 °C overnight. Reverse crosslinking of the protein–DNA complexes was performed according to the manufacturer’s protocol. Spin columns were used for ChIP DNA extraction. Standard PCR was performed, and products were detected via 3% agarose gel electrophoresis and visualized using a Syngene Bio Imaging system. The PCR primers used to amplify the promoter region are provided in Table 2. Potential binding sites (BS) for Sp1 within the promoter region of TFRC were identified using JASPAR (http://jaspar.genereg.net/).

The isolates were confirmed as C. jejuni or C. coli by using a multiplex PCR (mPCR) assay [17 (link)]. flaA-restriction fragment length polymorphism (RFLP) analysis was done as previously described [6 (link)]. The flaA amplicon was digested for 18 h at 37 °C with DdeI (Roche Diagnostics GmbH). The DNA segments were separated using 2.5 % agarose gels (Starlab GmbH, Hamburg, Germany) in tris-borate-EDTA buffer at 200 V for 1 h, stained with ethidium bromide and visualized under UV light. Documentation was done using a Bio Imaging System (Syngene, Cambridge, UK).

Sodium bisulfite–modified DNA was PCR amplified using two pairs of primers for each studied CpG site (one pair was specific for the methylated DNA, and the other for the unmethylated DNA) with EpiTect MSP Kit (Qiagen). β-tubulin-4 levels were used for normalization. Amplified products were analyzed by agarose gel electrophoresis. Agarose gel resolved products were analyzed using Gene Snap (Syngene) and Bio Imaging System (Syngene).