title: Sample 4
organism: Salmonella enterica subsp. enterica serovar Typhimurium
description: Protocols: Three-day-old alfalfa seedlings, grown on MS agar, were infected at the shoot-root junction with a 5 μL droplet of a S. Typhimurium transposon mutant library (described by Holden et al. (2022)), normalised to an OD600nm of 0.01 with 1 mM IPTG to induce transcription from the transposon-located promoter. Seedlings were then allowed to grow at 30 oC before sampling following 24, 48 and 72 hours growth. Ten seedlings were processed per timepoint and were homogenised in 1 mL of sterile PBS using a plastic pestle. Samples were filtered through 5 μm syringe filters to isolate bacterial cells and eliminate plant cell contamination. Alfalfa seeds were sterilised by immersion in a Falcon tube containing 20 mL of 70% ethanol for 30 seconds, followed by three sequential rinses with 20 mL sterile water. Subsequently, the seeds were treated with 5% sodium hypochlorite (20 mL) for 3 minutes on a rolling platform. Three subsequent washes in water were carried out. For germination, sterilised seeds were transferred to square agar plates (20 mL) containing Murashige-Skoog (MS) agar medium. These seeds were positioned with sufficient spacing to allow for three days of germination, reaching an approximate size of 1 cm. Following germination, the seedlings were transferred to fresh MS plates and infected with S. Typhimurium strain. Adequate seedlings were included in the process to enable replication for experimental purposes. Genomic DNA was extracted from these cells following the protocol described by Trampari et al. (2021). A Museek DNA fragment library preparation kit (ThermoFisher) was used to tagment genomic DNA and was then purified with AMPure XP beads (Beckman Coulter). DNA fragments were amplified using customised primers that anneal to the tagmented ends and biotinylated primers that anneal to the transposon. These PCR products were purified as previously and biotinylated DNA was incubated for 4 hours with streptavidin beads (Dynabeads® kilobaseBINDER™, Invitrogen) to capture only DNA fragments containing the transposon. These fragments were amplified using barcoded sequencing primers that anneal to the tagmented ends and to the transposon (Yasir et al., 2020). DNA fragments were then purified and size-selected using AMPure beads. Fragment length was quantified using a Tapestation (Aligent)