GSM5004391: delta_nuoBcydB_R2; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
3 mL of cell broth was taken at OD600~0.6 and immediately added to 2 volumes Qiagen RNAprotect Bacteria Reagent (6 mL). Then, the sample was vortexed for 5 seconds, incubated at room temperature for 5 minutes, and immediately centrifuged for 10 minutes at 5000 x g. The supernatant was decanted, and the cell pellet was stored in the -80°C. Cell pellets were thawed and incubated with Readylyse Lysozyme, SuperaseIn, Protease K, and 20% SDS for 20 minutes at 37°C. Total RNA was isolated and purified using the RNeasy Plus Mini Kit (Qiagen) columns following vendor procedures. An on-column DNase-treatment was performed for 30 minutes at room temperature. RNA was quantified using a Nano drop and quality assessed by running an RNA-nano chip on a bioanalyzer. The rRNA was removed using Illumina Ribo-Zero rRNA Removal Kit (Gram-Negative Bacteria). The quantity was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific). The quality was checked using RNA 6000 Pico Kit using Agilent 2100 Bioanalyzer (Agilent). Paired-end, strand-specific RNA-seq library was built with the KAPA RNA Hyper Prep kit (Kapa Biosystems) following manufacturer's instructions. RNA libraries were prepared for sequencing using standard Illumina protocols.