CD14+ Bovine Milk Macrophage Challenge with Streptococcus uberis

CD14+ Macrophage were challenged with Wildtype (WT), or S1154-deletion mutant (Del), and sequenced at timepoints zero hours (T0) and twenty-four hours (T24). To generate DEGs T0 and T24 timepoints were compared, a multifactor DESEQ2 run comparing T0 and T24 in both Wildtype and Deletion strains, and a direct comparison of T24 timepoints in Mutant and Deletion were generated. Overall design: Three replicates of each of: WT T0; WT T24; Del T0; Del T24 were generated using the Quantseq-3' FWD protocol, with the additional of the optional UMI during second strand synthesis. After sequencing across four lanes of a Nextseq 500 high yield run, the data was demulitplexed using BCL2FASTQ with the --no-lane-splitting option enabled. UMIs were used to correct for any PCR amplification errors during alignment using the standard Quantseq-3' FWD-UMI pipeline. For final analysis, (UMI-corrected) read counts were read into R studio and differential expression analysis carried out with DESEQ2. The various plots were generated in graphpad prism or R studio using the enhanced volcano and ggplot2 libraries.