Elucidation of the spatially regulated macrophage-induced transcriptional response of Yersinia pseudotuberculosis growing within synthetic microcolonies
217 ul of pre-heated (95 oC) Max Bacterial Enhancement reagent (Ambion #16122-012) was added to frozen bacterial pellets. Afterward, samples were transferred to a 1.5 ml microfuge tube and incubated at 95 oC for 5 minutes. Extraction of total RNA was conducted using the Zymo Direct-Zol Microprep Kit and following the protocol for cells in suspension (Zymo #R2026). cDNA libraries were generated following the RNAtag-seq protocol as previously described (Jensen et al., 2017; Shishkin et al., 2015), prokaryotic ribosomal RNA (rRNA) was depleted using riboPOOL pan-prokaryote following the manufacturers protocol (siTOOLS BIOTECH). Libraries were sequenced at high depth on Illumina.