title: Illumina HiSeq 1500 paired end sequencing; RNAseq of the the Growth Modulon of Corynebacterium glutamicum under Glucose Limited Chemostat Conditions
design: RNAseq of the the Growth Modulon of Corynebacterium glutamicum under Glucose Limited Chemostat Conditions
library_construction_protocol: Samples of 2 mL were withdrawn from the bioreactor and immediately centrifuged at 20000 g for 30 seconds at 4 °C. The supernatant was discarded and the remaining cell pellet was immediately flash frozen in liquid nitrogen. C. glutamicum ATCC 13032 growing under glucose limited chemostat conditions with growth rate = 0.2, 0.3, and 0.4 h-1. In detail: 2.2.1 Bioreactor setup Continuous chemostat processes with C. glutamicum were conducted in a 3 L steel bioreactor (KLF 2000, Bioengineering, Switzerland) equipped with process control (Lab view 2010, National Labs). Cooling and heating elements and a temperature sensor (Pt100, Bioengineering, Switzerland) ensured constant cultivation temperature of 30 °C. Mixing was realized with a six blade Rushton turbine. A pressure probe (PR-35 X HT, KELLER Druckmesstechnik, Jestetten, Germany) and an automatic exhaust valve were installed at the head of the reactor to regulate pressure (1.5 bar). For aeration, compressed air was channeled through a mass flow controller (MFC GFC171S, Analyt-MTC GmbH, Müllheim, Germany) and filtered aseptically (Midisart® 2000, Sartorius stedim biotech, Goettingen, Germany) before entering the bioreactor. Dissolved oxygen was monitored by an amperometric pO2 electrode (InPro 6800, Mettler-Toledo, Germany) and pH with a conventional pH probe (405-DPAS-SC-K8S, Mettler-Toledo, Germany). Molar fractions of oxygen and carbon dioxide in the exhaust gas were measured by a sensor (BCP-O2/CO2, BlueSens, Germany). To enable continuous processes in chemostat mode at fixed dilution rates (D, h-1) equaling the growth rate (µ, h-1; cf. section 2.5), the advanced controller scheme described in Graf and Haas et al. (2019) was employed. Thereby, a defined reaction volume in the bioreactor was maintained and constant feed and harvest rates were ensured. Prior to all cultivations, the reactor was sterilized with 1 mol L-1 K2HPO4/KH2HPO4 buffer (pH 7) for 20 min at 121 °C. Prior removal of the buffer, the drain of the reactor was sterilized in situ with pure steam for 10 min.  Total RNA was isolated from three biological replicates using RNeasy Mini Kit along with a DNase Kit (both from Qiagen).  Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to prepare cDNA libraries.