Nanodrop spectrophotometer

Three 120 mL main cultures of D. shibaeΔluxI₁ were inoculated from precultures as described above. After reaching the desired OD₆₀₀ cultures were split into two cultures (induced and non-induced control) and incubated as described above. Before the split and at time points 10, 20, 40, 60, 120, and 180 min post-induction, two samples of 2 mL per treatment were withdrawn for transcriptomics and processed immediately. Additionally, 1 mL samples were withdrawn for flow cytometric analysis. The transcriptome samples were centrifuged (13,000 rpm, 1 min, room temperature), the supernatant was removed, and 500 μL Trizol reagent (Ambion, Life Technologies, Carlsbad, CA, United States) was added. Samples were snap frozen using liquid nitrogen and stored at -70°C. Isolation of RNA was performed as described (Wang et al., 2014a (link)). RNA amount was determined using the NanoDrop spectrophotometer (Peqlab, Erlangen, Germany). The following samples were sequenced in three biological replicates: pre-induction, 10, 20, 40, 60, 120, and 180 min post-induction with AHL, 60 and 180 min after addition of the solvent DMSO (control).

Cells were collected by centrifugation at 12,000 × g for 1 min at 4°C, covered with 1 ml Trizol reagent (Ambion, Germany), immediately frozen in liquid N₂ and stored at -70°C until processing. For RNA extraction, cells were homogenized with ~ 0.3 g of glass beads in the FastPrep-24 instrument (MP Biomedicals, California, USA) at 6.0 m/s for 3 min and then incubated for 5 min at room temperature. Samples were centrifuged at 12,000 × g for 10 min at 4°C and the supernatants were transferred to fresh tubes, followed by the addition of 100 μl of 1-bromo-3-chloropropane (BCP, Sigma, Germany) and incubation for 10 min at room temperature. Samples were centrifuged at 12,000 × g for 10 min at 4°C, after which the aqueous phase was transferred to new tubes and mixed with 500 μl of absolute ethanol. Extracts were applied to RNeasy spin columns (RNeasy mini kit, Qiagen, Hilden, Germany) and processed according to the manufacturer’s instructions. In addition, samples were treated with DNAse I (Qiagen, Hilden, Germany). Removal of genomic DNA was verified via PCR. The concentration of the RNA was quantified using a NanoDrop spectrophotometer (Peqlab, Erlangen, Germany) and the RNA integrity was assessed using Bioanalyzer 2100 (Agilent, Santa clara, USA).

Amounts of CTCs in blood and DTCs in the right lung and bone marrow were determined by a real-time polymerase chain reaction [42 (link),52 (link),53 (link)]. DNA concentrations of all samples were quantified using a NanoDrop spectrophotometer (Peqlab, Erlangen, Germany). QPCR was performed with established human-specific Alu-primers [42 (link),52 (link),53 (link)]. A total of 2 μL total DNA (60 ng lung/bone marrow-DNA; 20 ng blood-DNA) was used for each qPCR. Numerical data were determined against a standard curve, as previously described [54 (link)].

Tissue samples from the left hemisphere were taken via micropunches of 1 mm diameter from the mPFC, M1 and OFC. Further tissue samples were taken in the same way from both hemispheres from the CPu. Tissue was homogenized by ultrasonication in the buffer provided by the NucleoSpin RNA/Protein-Kit (Machery-Nagel, Düven, Germany). The total RNA and protein was extracted as recommended in its user manual. RNA concentrations were determined using a Nanodrop Spectrophotometer (peqlab). cDNA was synthesized using the High Capacity RNA-to-cDNA Kit (Lifetechnologies). TaqMan qPCR was performed with StepOne Real-Time PCR System (Lifetechnologies) using TaqMan fast advanced master mix (Lifetechnologies). The following TaqMan Gene Expression assays (Lifetechnologies) were used: Pvalb Assay (Rn00574541_m1) and c-Fos Assay (Mm00487425_m1). CT values were normalized to the housekeeping gene GFAP (Rn01253033_m1). Fold change was calculated using the ∆∆CT method.

Trichoderma reesei QM9414, QM9978, and Rut-C30 were used for the generation of vib1 deletion and overexpression strains. Protoplast preparation and transformation were performed as previously described [69 (link)]. The deletion cassettes were purified after PCR (QIAquick PCR Purification kit, QIAGEN) and concentrations were determined (Nanodrop Spectrophotometer, Peqlab). After transformation protoplasts were stabilized and regenerated on selection medium supplemented with 100 mg/mL hygromycin B. For sporulation, transformants were transferred to 12-well plates and purified by plating conidiospores onto plates with 0.1% Triton X-100 as colony restrictor. Single colonies were transferred to selective medium and screened for correct integration of the deletion or overexpression cassettes by PCR analysis (primer abbreviated with “ch” in Additional file 3: Table S2). Insertion of the gpd-vib1 overexpression cassette was verified with primer pairs TR46-verif 5′vib1 for + TR47-verif 5′vib1 rev, TR48-verif 3′ vib1 for + TR49-verif 3′vib1 rev, and TR50-verif hph vib1 for + TR51-verif hph vib1 rev amplifying the 5′or 3′ region and the hph marker gene, respectively. Three or four independent clones were isolated for each strain construction and analyzed as biological replicates.

mIMCD3 cells were seeded in 12 well plates, treated with DMSO (AppliChem), 4 ng/100 µl TNFα (aa80–235; R&D), 2 µg/100 µl cycloheximide (C4859; Sigma) for 16 h and washed with PBS right before lysis in Tri-Reagent (Sigma). For RNA isolation from kidney tissue, one-quarter of a kidney was ground with BeadBeater (Roth) using a Precelly24 with 5000 rpm two times for 30 s in Tri-Reagent. RNA extraction was performed with the Direct-zol RNA Miniprep kit (Zymo Research) following the manufacturer’s instructions, including a DNase1 treatment step. Prior to the reverse transcription by using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems), RNA concentration and sample quality were assessed on a Nanodrop spectrophotometer (Peqlab). mRNA was assessed by SYBR Green (ThermoFisher Scientific) qPCR using mHprt1 as endogenous control. Primers are listed in Supplementary Table S1. The qPCR experiments were performed on a QuantStudio 12 K Flex Real-time PCR System (ThermoFisher Scientific). For data analysis, all results were normalized to the housekeeping gene Hphrt1 using the delta-delta CT followed by a two-tailed Student’s t test (p < 0.05).