LoVo cells were maintained in Kaign's modification of Ham's F12 medium (F12-K) supplemented with 10% (vol/vol) heat-inactivated foetal bovine serum (FBS). The culture medium was changed every 24-h, and kept for further use during the infection as ”conditioned medium”. Listeria monocytonenes (Lm, LL195 strain) was grown in Brain Heart Infusion medium (BHI) at 37°C to early saturation, washed in 37°C PBS and diluted in conditioned medium. LoVo cells were plated in 75 cm2 culture flasks 4 days before infection and, upon reaching 80% confluence, Lm was added at a multiplicity of infection (MOI) of 30 colony forming unit (CFU)/cell in conditioned medium. Flasks were then centrifuged at 200 x g for 2 minutes, and incubated at 37°C and 5% CO2 for 30 min. After 30min, the inoculum was removed and the cells were incubated in conditioned medium containing 25 µg/ml gentamicin, at 37°C until 5 hours post-infection. Cells were rinsed with 5 ml of ice-cold PBS(100µg/ml cycloheximide) twice. The PBS was thoroughly aspirated and the T75 flaks were submerged in a reservoir of liquid nitrogen for 10 s, and stored at -80°C until lysis. To proceed with lysis, flasks were transferred to dry ice and 200 µl of lysis buffer was added per flask [20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml cycloheximide and 25 U/ml TurboDNase (ThermoFisher Scientific)]. Cells were scraped extensively to ensure lysis after which the lysate was incubated on ice for 10 minutes and then triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 10 min at 21,000 g at 4°C. The supernatants were recovered separated in two fractions: 350 µl was used for RNase footprinting for RiboSeq based on the original protocols of Ingolia and colleagues (Ingolia et al., 2012 doi:10.1038/nprot.2012.086), while 50 µl was directly used in RNA extraction using acid-phenol chloroform.