Rnase free dnase 1 treatment

From overnight grown cultures of S. Enteritidis WT and ΔSEN1005 strains, subculture was done in SPI-1 inducing media to reach O.D₆₀₀ at ∼0.6. For RNA sequencing, the subcultured bacteria were snap-frozen in liquid nitrogen before RNA isolation. RNA was extracted using Trizol reagent (Ambion), followed by RNase free DNaseI treatment (Thermo Fisher Scientific) and cDNA synthesis using kit from HIMedia. With the diluted and normalized cDNA templates, quantitative RT PCR was performed using Kapa Sybr Fast qPCR Master Mix (2x; Kapa Biosystems, USA). Either 16s rRNA or Guanylate monophosphate kinase (gmK) genes was used as housekeeping genes for the bacterial samples and gapdh for mammalian samples in the qRT-PCR assays. The qRT-PCR data were presented corresponding to the fold-change differences in gene expression in ΔSEN1005 with respect to WT. Experiments were repeated thrice in triplicates. All the primers used are listed in supplementary Table S2.
For infection experiments, a MOI of 50 bacteria/cell was used and after 2 hours p.i. the mammalian cells were harvested and immediately processed for RNA extraction, subsequent purification, cDNA synthesis and qRT-PCR analysis as already discussed.

Bacterial DNA was extracted using a QIAprep mini-spin kit (Qiagen, Valencia, CA) following the manufacturer’s protocol specifications. Purified DNA was quantified by measuring absorbance at an optical density of 260 nm (OD₂₆₀) and OD₂₈₀. Bacterial RNA was extracted following cultivation in either ambient air or 5% CO₂ using TRIzol reagent followed by RNase-free DNase I treatment (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s specifications.

RAW 264.7 cells were treated with LPS as described in the earlier sections (6×10⁵ in a six well plate and stimulated with 50ng/ml LPS along with different concentrations of BSE). After 16h of treatment, total cellular RNA was isolated using Trizol reagent^® (Ambion, Life Technologies), followed by RNase-free DNase I treatment (Thermo Fisher Scientific) to remove any genomic DNA. One microgram of total RNA was reverse transcribed into cDNA using the revert-aid first strand cDNA synthesis kit (Thermo Fisher Scientific), and quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green qPCR master mix (Thermo Scientific) using Light cycler 96 (Roche Life Science). β-actin gene expression was used as housekeeping control. The following primers were used for the analysis.