Rdnase set

mRNA was extracted from snap‐frozen 16h‐stimulated moDCs using a NucleoSpin® RNA Plus kit (Macherey‐Nagel, Germany) in combination with the rDNAse set (Macherey‐Nagel, Germany) to remove contaminating DNA. cDNA was synthesized using an iScript™ advanced kit (Bio‐Rad, USA) according to the manufacturer's protocol in the PTC‐100 Programmable Thermal Controller (MJ research, PTC‐100). cDNA and mRNA samples were stored at ‐80°C. Quantitative analysis was conducted on a CFX96 Real‐Time C1000 Thermal Cycler detection system with the use of an IQ™ SYBR® Green Supermix, according to manufacturer's protocol (both from Bio‐Rad). Primers for the genes of interest are listed in Table 1. Primers (produced by Biolegend, USA) were designed with Primerblast (NCBI, USA) and Beacon Designer (Premier Biosoft International, USA). Primers were validated based on the efficiency and R². Primer specificity was tested by running the products on a 2% agarose gel (70 minutes; 110V). The expression of the genes of interest was determined using 12.5ng cDNA and SYBR green (Bio‐rad, USA). The RT‐PCR protocol consists of 3 min at 95°C followed by 10 s at 95°C and 30 s at the optimal primer annealing temperature as determined beforehand. The samples were measured in duplo and mean Ct values were used to determine the relative expression using the Pflaff method and β‐actin as a reference gene.

For transcript analysis, 2 × 1 mL samples from Actinoplanes cultures were taken during growth phase, separated from the supernatant by centrifugation (10 s) and snap-frozen in liquid nitrogen. Pellets were stored at − 80 °C until further processing. Cell disruption, RNA isolation, and digestion of DNA from frozen cell pellets were performed by use of 2-mL lysing matrix tubes (0.1 mm spherical silica beads, MP Biomedicals, Santa Ana, California, USA) and the NucleoSpin® RNA Plus kit in combination with the rDNAse Set (Macherey-Nagel, Düren, Germany) according to a protocol of Schaffert et al. (2019a ). Residual DNA was tested negatively with two primer pairs binding to genomic DNA of Actinoplanes sp. SE50/110 and amplifying small fragments at round about 200–300 nt. The quantity of RNA was analyzed with the NanoDrop 1000 spectrometer (Peqlab, Erlangen, Germany).

RNA was extracted using the Rneasy plant mini extraction kit (Qiagen). The plant material in RNAlater was thawed just enough to remove approximately 20–30 mg. This was placed into a tube with a sterile stainless steel 6 mm bead. The tube, sample, bead and adapter set were then cooled at −80 °C for 30 min. After cooling, the plant tissue was disrupted by beating using the TissueLyser II (Qiagen) at 30 Hz for 2 min. The kit protocol was then followed for the remainder of the extraction. The RNA was eluted in 50 µl of sterile water. Extractions were pooled and concentrated as described above. Before concentrating, the pooled RNA was treated with DNase, using the rDNase Set (Macherey-Nagel).