GSM9030766: Inoculum_biorep_3; Acinetobacter baumannii; RNA-Seq
Total RNA from infected cells, non-infected cells and ABC141 inocula was extracted following a previously described method (Kröger et al., 2018). For cultures, bacteria were lysed with 2/5 volume of ice-cold 5% (v/v) phenol, 95% (v/v) ethanol ultra-pure solution for 1 h on ice. The same lysis process was applied to the total of infected cells and mock-infected cells sorted by FACS. A centrifugation at 3220 x g for 10 min at 4°C pelleted the sample, which was resuspended with 1 mL ice-cold TRIzol and transferred into a Phase-Lock-Tube (VWR, #2302830). 400 µL of chloroform was added and the solution was shaken (without vortexing) for 10 s, followed by a 3 min incubation at RT. The tubes were centrifuged at 12000 x g at 4 °C for 15 min. Then, the aqueous phase was transferred to a new 1.5 mL tube. 450 µL of isopropanol was added and mixed before a 30 min incubation at RT. A new centrifugation at full speed for 30 min preceded three washes of the pellet with 350 µL of ethanol 75%, a 10 min incubation at RT and a 10 min centrifugation at full speed. The last supernatant was discarded and the pellet air dried before resuspension with 25 µL of RNAse-free water, followed by a 5 min incubation at 65 °C at 900 rpm. The RNA extraction quality was verified with a 1/10 dilution using the 4200 TapeStation (Agilent Technologies). Total RNA was sent to Core Unit Systems Medicine (SysMed) sequencing facility for RNA-seq (University of Würzburg, Germany), where DNase I digestion and rRNA depletion (RiboCop) was performed. Library for the sequencing was prepared using the NEBNext Multiplex Small RNA Library Prep kit. Sequencing was performed on the Illumina NextSeq-500 Mid Output KT v2.5 (150 cycles).