Illumina HiSeq 1500 sequencing; The transcription dynamics in Actinoplanes sp. SE50/110 during growth.
The transcription dynamics in Actinoplanes sp. SE50/110 during growth.
Samples were collected by centrifugation of 1 mL of bacterial culture for 15 sec at maximum speed. Supernatant was discarded and pellets were subsequently froozen in liquid nitrogen.  RNA was isolated with a Macherey-Nagel NucleoSpin® RNA Plus Kit (MACHEREY-NAGEL, Düren, Germany) in combination with an rDNase Set (MACHEREY-NAGEL, Düren, Germany) as described previously (Schaffert et al., 2019a). In brief, frozen cell pellets were resuspended in 500 µL LB-buffer and transferred to 2 mL lysing matrix tubes. Cell disruption was carried out in a homogenizer three times for 20 s at 6.5 m/s and cooled on ice between steps. Subsequently, the cell suspension was centrifuged, and the RNA was extracted from the supernatant using the NucleoSpin® RNA Plus Kit in combination with rDNase Set for an on-column DNA digestion. After clean-up the DNA-digestion was repeated in-solution. Residual DNA was tested negatively with two primer pairs binding to genomic DNA of Actinoplanes sp. SE50/110. Quality and quantity of the RNA were analyzed with an Xpose® spectrophotometer (Unchained Labs, Pleasanton, CA, USA) and an Agilent RNA 6000 Pico kit run on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).  For each time point the RNA of three fermenters was isolated and used for cDNA library preparation. The preparation of cDNA libraries were performed according to Wolf et al., 2017b. Additionally, a pool was generated for each fermenter of all time points using equimolar amount of RNA.
Experimental Factor: growth condition
transition phase
Experimental Factor: time
120