GSM7035081: menF KO adapted 2, rep 1; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
3 ml of cell broth (at an A600 ≈ 0.6) was immediately added to two volumes of Qiagen RNA-protect Bacteria Reagent (6 ml), vortexed for 5 s, incubated at room temperature for 5 min and immediately centrifuged for 10 min at 17,500 r.p.m. The supernatant was decanted, and the cell pellet was stored at −80 °C. Cell pellets were thawed and incubated with Ready-Lyse Lysozyme, SuperaseIn, protease K and 20% SDS for 20 min at 37 °C. Total RNA was isolated and purified using the Qiagen RNeasy Mini Kit columns and following vendor procedures. An on-column DNase treatment was performed for 30 min at room temperature. RNA was quantified using a Nano drop and quality assessed by running an RNA nano chip on a bioanalyser. The ribosomal RNA was removed using an Illumina Ribo-Zero rRNA removal kit for Gram-negative bacteria. A KAPA Stranded RNA-Seq Kit (Kapa Biosystems KK8401) was used following the manufacturer's protocol to create sequencing libraries with an average insert length of around 300 bp