Nanodrop 1000 spectrometer

For transcriptome analysis, 2 × 1 mL samples from Actinoplanes culture 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.
For RNA isolation, frozen cell pellets were resuspended in 500 μL LB-buffer (NucleoSpin^® RNA Plus, Macherey–Nagel, Düren, Germany) and transferred to 2 mL lysing matrix tubes (0.1 mm spherical silica beads, MP Biomedicals, Santa Ana, California, USA). Cell disruption was carried out in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) for three times 20 s at speed setting 6.5 and 5 min on ice in between. Subsequently, the cell suspension was centrifuged for 5 min at 13,000×g and 4 °C. The supernatant was used for RNA extraction using the NucleoSpin^® RNA Plus Kit in combination with rDNase Set (Macherey-Nagel, Düren, Germany) for an on-column DNA digestion. After clean-up and elution according to manufacturer’s protocol, the DNA-digestion was repeated in-solution and the sample cleaned up again by use of the same kit. 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).

For the transcriptome analysis, RNA was isolated using a Macherey-Nagel NucleoSpin® RNA Plus kit in combination with Macherey-Nagel rDNase Set (Macherey-Nagel, Düren, Germany). Therefore, cell pellets were resuspended in 500 μL LBP buffer (NucleoSpin® RNA Plus kit, Macherey-Nagel) and transferred into 2-mL lysing matrix tubes (0.1-mm spherical silica beads, MP Biomedicals, Santa Ana, CA, USA). Cell disruption was carried out in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) two times for 30 s at speed setting 6.5 and 1 min on ice in between. Following this, cell debris were centrifuged for 2 min at maximum speed at 4 °C. The supernatant was used for RNA isolation according to the manufacturer’s protocol. To verify the complete removal of residual DNA in the samples, PCR with primers binding to genomic Actinoplanes sp. SE50/110 DNA was performed. Quality and quantity of the RNA were analyzed with a NanoDrop 1000 spectrometer (Peqlab, Erlangen, Germany) and an Agilent RNA 6000 Pico kit run on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).

RNA was prepared using RNeasy Micro Kit (Qiagen). RNA quantity and quality was assessed using a Nanodrop 1000 Spectrometer (Peqlab, Erlangen, Germany) and Agilent 2100 Bioanalyzer. From in vitro-generated cells (M1-, M2-macrophages) and CD14⁺ monocytes, three replica pools (each consisting of RNA from 5 different HD) were generated. RNA from flow-sorted cells was not pooled (Table S2). RNA (30 ng of each replica pool; 0.5–15 ng of sorted cells) was subsequently amplified and converted into cDNA by a linear amplification method using WT-Ovation PicoSL System in combination with the Encore^® Biotin Module (both from NuGen, San Carlos, CA, USA). cDNA was hybridized to Affymetrix GeneChip^® Human Gene 1.0 ST Arrays.

For RNA isolation frozen cell pellets were suspended in 800 μL RLT buffer (RNeasy mini kit, Qiagen, Hilden, Germany) and transferred to 2 mL lysing matrix tubes (0.1 mm spherical silica beads, MP Biomedicals, Santa Ana, California, USA). Cell disruption was carried out in a homogenizer (FastPrep FP120, Thermo Fisher Scientific, Waltham, MA, USA) for two times 20 s at speed setting 6.5 and 1 min on ice in between. Subsequently, the cell suspension was centrifuged for 3 min at 13,000 g and 4 °C. The supernatant was used for RNA extraction using a Qiagen RNeasy mini kit in combination with an RNase-free DNase kit (Qiagen, Hilden, Germany) for on-column and off-column DNA digestion. PCR with primers binding to genomic Actinoplanes sp. SE50/110 DNA was used to verify complete removal of residual DNA. Quality and quantity of the RNA was analyzed with a NanoDrop 1000 spectrometer (Peqlab, Erlangen, Germany) and an Agilent RNA 6000 Pico kit run on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).