GSM8479020: HexR knockout, M9 succinate, bioreplicate 2; Pseudomonas putida; RNA-Seq
Cell culture harvested at the mid–log phase (2 ml) was mixed with 4 ml of RNAprotect® Bacteria Reagent (Qiagen, Valencia, CA, USA) by vortexing for 5 seconds, incubated at room temperature for 20 minutes, then centrifuged at 5000×g for 10 minutes. The supernatant was decanted, and any residual supernatant was removed by inverting the tube onto a paper towel. Total RNA samples were isolated using RNeasy® Plus Mini Kit (Qiagen) according to the manufacturer's instructions. Samples were then quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). The RNA quality was checked using RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer (Agilent). The ribosomal RNAs (rRNAs) were removed from 2 μg of the total RNA using riboPooL Probes (Pan–prokaryote) siTOOLS BIOTECH and Dynabeads™ MyOne Streptavidin C1 (Invitrogen #65001). RNA samples were subjected to further purification and Dnase treatment using the RNA clean & concentrator kit (Zymoresearch R1013). Paired–end, strand–specific RNA–seq library was built from the rRNA–depleted RNA sample using the KAPA Stranded RNA–Seq Library Preparation Kit (KAPA Biosystems). The obtained libraries were analyzed on Agilent Bioanalyzer DNA 1000 chip (Agilent) and sequenced using NextSeq 500/550 or HiSeq X (Illumina). Two replicates were obtained for each sample