Fastprep 24 classic instrument

To compare the expression of coloration and pigment genes between melanic bars and interbars regions, we sampled the whole melanic and non-melanic skin region. Skin tissue was dissected and kept in RNAlater (Invitrogen) at 4°C overnight and then transferred to −20°C for long-term storage. RNAlater was removed prior to homogenization. Skin samples and the appropriate amount of TRIzol (Invitrogen) (1 ml TRIzol per 100 mg sample) were homogenized in 2 ml Lysing Matrix A tube (MP Biomedicals) using FastPrep-24 Classic Instrument (MP Biomedicals). RNA was extracted according to the manufacturer’s recommendations (Invitrogen) with an additional wash step by 75% Ethanol. Subsequent purification and on-column DNase treatment were performed with the RNeasy Mini Kit (Qiagen) and RNase-Free DNase Set (Qiagen). Following extraction and purification, RNA was quantified using Qubit RNA BR Assay Kit (Invitrogen) with Qubit Fluorometer (Life Technologies).

Faecal samples were defrosted and mixed with 400 μL of phosphate buffer (pH 7.4, 100% D₂O, 3 mM of NaN₃, 1 mM of 3-(trimethyl-silyl)-[2,2,3,3-²H4]-propionic acid (TSP) for the chemical shift reference at δ0.0) and Zirconium beads (0.45 g ±0.1). The samples were vortexed and then homogenised with a FastPrep-24 Classic Instrument (MP BIOMEDICALS) (30 sec per cycle, speed 6.0, 2 cycles). After a centrifugation (13,000 xg, 15 min), 180 μL of the supernatants were collected and transferred in 3-mm tubes for ¹H nuclear magnetic resonance (NMR) spectroscopic analysis, which was performed on a Bruker 600 MHz spectrometer (Bruker Biospin, Karlsruhe, Germany) at 300K (26.85°). The parameters of the acquisition were as previously reported for urine[61 (link)]. 4 dummy scans followed per 64 scans were acquired for each spectrum which were then imported into Matlab (version R2014a, The Mathworks Inc.). The region δ4.82–4.76 was removed to eliminate residual water signal. All spectra were normalised according probabilistic quotient method and automatically aligned. Principal Components Analysis (PCA) was performed with mean-centring and Pareto scaling.

Plasmid DNA was extracted using the PeqGOLD plasmid miniprep kit (VWR International GmbH, Darmstadt, Germany) according to the manufacturer’s manual.
F. culmorum was grown in PDB at room temperature with constant shaking for 3 days. Mycelia were collected, and DNA was isolated using the PeqGOLD fungal DNA mini kit (VWR International GmbH, Darmstadt, Germany) according to the manufacturer’s manual. Alternatively, DNA was extracted from fungal mycelium collected from PDA plates, in accordance with Liu et al. [41 (link)].
Fungal mycelia, as well as wheat leaves, were homogenized using a FastPrep-24™ classic instrument (MP Biomedicals GmbH, Eschwege, Germany) with liquid nitrogen and sterile steel spheres for RNA extraction. RNA isolation was performed using a plant RNA isolation mini kit (Agilent Technologies, Inc., Wilmington, NC, USA.) according to the manufacturer’s manual.

For RNA sample collection, exponentially growing cultures at 30 and 37°C were divided into two, with one exposed to 0.6 mW cm⁻² and one in darkness, and kept at the same temperature at which they had been growing. Samples were collected at 10 min intervals and stabilized in RNALater (Sigma). RNA was extracted using the RNeasy mini kit (Qiagen) according to manufacturers’ instructions. Cells were lysed by bead beating for two 40 s cycles at 6 m/s in FastPrep® 1 ml Matrix B lysis tubes (MP Biomedicals) with a FastPrep®-24 Classic Instrument (MP Biomedicals). The RNA was quantified by NanoDrop and contaminating DNA was removed using TURBO DNA-free (Ambion) according to manufacturers’ instructions. RNA was quantified and the RNA Integrity was measured using the Bioanalyzer (Agilent) according to manufacturers’ instructions. Only RNA with a RIN of greater than 8 was accepted for conversion to cDNA. For cDNA generation, 15 μl of RNA was added to 1 μl 10 mM dNTPs (Sigma) and 1 μl random primers (Invitrogen) and incubated 5 min and 65°C then on ice for 1 min. The sample was centrifuged 5 s and 4 μl first strand buffer, 1 μl 0.1 M DTT, and 1 μl Superscript III (all Invitrogen) was added. The sample was incubated for 5 min at 25°C, 60 min at 50°C, and 15 min at 70°C and then stored immediately at −80°C.

Cells were collected and washed with 100 mM Tris–HCl buffer (pH 7.5). Mechanical lysis of cells was performed with glass beads using FastPrep^®‐24 Classic Instrument (MP Biomedicals, USA). The crude extract was centrifuged at 12,000g for 10 min at 4 ℃, and the supernatant was used for enzyme activity assay. The protein concentration was determined with BCA Protein Assay Kit (Thermo Fisher Scientific, USA). ALAS activity was determined by measuring 5-ALA formation (µmol/L min) [25 (link)]. The reaction mixture contained 100 mM Tris–HCl (pH 7.5), 200 mM glycine, 0.2 mM succinyl-CoA, 0.1 mM pyridoxal phosphate (PLP) and 20 µg crude extract. After proceeding at 37 ℃ for 10 min, the reaction was terminated by the addition of 10% (v/v) trichloroacetic acid. Concentration of 5-ALA in the supernatant was determined. PPC activity was analyzed by a coupled reaction with malate dehydrogenase at 30 °C as previously described [54 ]. The reaction mixture contained 100 mM Tris–HCl (pH 7.5), 2 mM phosphoenolpyruvate, 10 mM NaHCO₃, 10 mM MnSO₄, 0.1 mM NADH, 20% (v/v) glycerol, 1.6 U malate dehydrogenase, and 20 µg crude extract. The activity was assayed spectrophotometrically by monitoring the decrease in absorbance of NADH at 340 nm.

Mock-infected (age-matched) and 22L-infected mouse brain homogenates (10% w/v) at the terminal stages of prion disease (stored at −80 °C) were prepared in lysis buffer (50 mM Tris-Cl [pH 7.5], 250 mM NaCl, 0.5% Triton X-100 and protease inhibitor cocktail [1×]) using a mechanical homogenizer (MP Biomedicals FastPrep-24 Classic Instrument [MP Biomedicals]). The lysates were centrifuged at 3000 g, and the supernatant was collected and subjected to Pierce bicinchoninic acid assay for protein quantification. Equal amounts of protein were subjected to the immune pull-down assay by overnight incubation with 2 μg of anti-active Rab7 antibody in lysis buffer in the presence of protease inhibitor cocktail. Following this, it was incubated with protein A Sepharose beads (GE Healthcare) for 3 h at 4 °C with rotation. The beads were washed with lysis buffer 5 times by centrifugation followed by the removal of supernatant prior to the final sample preparation by boiling the sample in 80 μl of 3× Laemmli buffer for 10 min. The samples were then subjected to further studies by immunoblotting.