Total genomic DNA and RNA were extracted from ~0.25 g of soil with slight modifications as described earlier (Männistö et al. 2016 ) using a CTAB-based method by Griffiths et al. (2000 ). Three replicate extractions were processed from each of the eight plots. Hexadecyltrimethylammoniumbromide (CTAB; 650 µl) extraction buffer and phenol–chloroform–isoamyl alcohol (25:24:1; pH 8.0; 650 µl) were added together with a mixture of beads to the sample tubes followed by bead beating on a Precellys 24 Dual homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) for 30 s at 5500 r m−1. The bead mixture contained 0.1 mm glass beads (0.3 g), 1.0 mm ceramic beads (0.7 g), and two large (3.5 mm) glass beads (Bio Spec Products Inc., Bartlesville, OK, USA). Samples were further processed as described in Männistö et al. (2016 ). DNA samples were treated with RNAse A (Thermo Scientific, Waltham, MA, USA) and RNA samples with DNAse I (Thermo Scientific) and converted to cDNA using the Revert Aid H Minus First Strand cDNA Synthesis kit (Thermo Scientific). All solutions used for RNA extraction were treated with 0.1% diethylpyrocarbonate. RNA and DNA concentrations were measured using a Qubit fluorometer and Quant-iT RNA and dsDNA HS assay kits (Thermo Scientific), respectively.
Glass bead
Manufactured by Biospec
Sourced in United States, Germany
Glass beads are small, spherical objects made of glass. They are used in various laboratory applications to facilitate mixing, stirring, and agitation of samples or solutions. Glass beads provide a smooth, inert surface and can be used to promote efficient mixing and homogenization in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fastprep 24, by MP Biomedicals (7 mentions)
Zirconia silica bead, by Biospec (6 mentions)
Mini beadbeater, by Biospec (5 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Mini beadbeater 16, by Biospec (3 mentions)
Rnalater, by Thermo Fisher Scientific (3 mentions)
Psp spin stool kit, by Stratec (3 mentions)
Magna lyser, by Roche (3 mentions)
Fastprep machine, by MP Biomedicals (3 mentions)
Gfp trap agarose beads, by Proteintech (3 mentions)
Alexa 680 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (3 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (3 mentions)
Tissuelyser, by Qiagen (3 mentions)
Rlt buffer, by Qiagen (3 mentions)
Sterile polypropylene microvials, by Biospec (2 mentions)
Inhibitex buffer, by Qiagen (2 mentions)
Qiaamp fast dna stool mini kit, by Qiagen (2 mentions)
110 protocols using glass bead
1
Soil Metagenome Extraction and Analysis
2
Microbiome Sampling from Mouse Stool
3
Microbiome Sampling from Mouse Stool
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To avoid the confounding effects of co-housing on the diversity of the intestinal microbiota for each treatment group we collected samples from mice housed in different cages. Fresh stool pellets were collected and stored at –80 °C. Frozen stool samples (~100 mg) were placed in sterile polypropylene microvials (BioSpec Products) containing 1 ml InhibitEX Buffer (QIAGEN), 1 ml of 0.1 mm diameter zirconia/silica beads (BioSpec Products) and one 3.5 mm diameter glass bead (Biospec Products). Samples were homogenized for 2 min using a Mini-BeadBeater 16 (BioSpec Products). DNA was purified with the QIAamp Fast DNA Stool Mini Kit (QIAGEN) according to the manufacturer’s instructions.
4
DNA Extraction from Cecal Contents
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DNA was extracted, from 0.25 g cecal content, using 700 μl Stool Transport and Recovery (STAR) buffer (Roche Diagnostics Nederland BV, the Netherlands). The cecal sample was transferred to a sterile screw-capped 2 ml tube (BIOplastics BV, the Netherlands) containing 0.5 g of zirconium beads (0.1 mm; BioSpec Products Inc., USA) and 5 glass beads (2.5 mm; BioSpec Products Inc., USA). The samples were treated in a bead beater (Precellys 24, Bertin technologies, France) at a speed of 5.5 ms− 1 for 3 × 1 min, followed by incubation at 95 °C with agitation (15 min and 300 rpm). The lysis tube was centrifuged (13,000 g for 5 min at 4 °C), and the supernatant was transferred to a 2 ml microcentrifuge tube. Thereafter, the above described process was repeated with 300 μl of STAR buffer. An aliquot (250 μL) of the combined supernatants from the sample lysis was then transferred into the custom Maxwell® 16 Tissue LEV Total RNA Purification Kit cartridge. The remainder of the extraction protocol was then carried out in the Maxwell® 16 Instrument (Promega, the Netherlands) according to the manufacturer’s instructions. DNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop® Technologies, USA), and DNA was stored at − 20 °C until further use.
5
Total RNA Extraction and cDNA Synthesis
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The cells were grown to an OD of 0.3–0.4 in CTFUD medium, centrifuged at 4 °C for 5 min, and immediately flash frozen in liquid N2. Pelleted cells were resuspended in 1.5 mL of TRIzol (Invitrogen, Carlsbad, CA). Glass beads (0.8 g of 0.1 mm Glass beads; BioSpec Products, Bartlesville, OK) were added to the cell suspension and lysed with 3 × 20 s bead beating treatments at 6500 rpm in a Precellys 24 high-throughput tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was purified using an RNeasy kit (Qiagen, Valencia, CA) with DNase I on-column treatment. RNA quantity was determined by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and RNA quality was assessed with Agilent Bioanalyzer (Agilent Technologies). RNA (10 µg) was used as the template to generate ds-cDNA using Invitrogen ds-cDNA synthesis kit according to the manufacturer’s protocols (Invitrogen).
6
Quantification of Ethanol in Paracoccidioides Cells
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The concentration of ethanol was quantified by enzymatic detection kit according to the manufacturer's instruction (UV-test for ethanol, RBiopharm, Darmstadt, Germany). Ethanol is oxidized to acetaldehyde by the enzyme alcohol dehydrogenase, in the presence of nicotinamide-adenine dinucleotide (NAD). Acetaldehyde is quantitatively oxidized to acetic acid in the presence of aldehyde dehydrogenase, releasing NADH, which is determined by means of its absorbance at 340 nm. Paracoccidioides Pb01 yeast cells were subjected or not to carbon starvation, and 106 cells were used to assay. Briefly, cells were counted, centrifuged, and lysed using glass beads and bead beater apparatus (BioSpec, Oklahoma, USA) in 5 cycles of 30 sec, keeping the samples on ice. The cell lysate was centrifuged at 10,000× g for 15 min at 4°C and the supernatant was used for enzymatic assay according to the manufacturer's instructions. The concentrations of ethanol were obtained in triplicate.
7
Rapid RNA Isolation from Yeast Cells
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Ten milliliters of yeast cells growing in YPD (yeast extract, peptone, dextrose) or synthetic dextrose (SD) media lacking the corresponding amino acid were harvested at OD 2 for RNA isolation. RNA was isolated using TRIzol (Invitrogen). Briefly, 100 µL worth 0.5-mm acid washed glass beads from BioSpec products (cat. no. 11079105) were added to yeast cell pellets in 2 mL screw cap tubes. One milliliter of TRIzol was added, and the tubes were vigorously vortexed using beadbeater (30 sec, four times on ice). The lysates were spun at 15,600g for 5 min and the supernatants were added to 0.2 volume of chloroform. After vigorous vortexing (>1 min) and phase separation, the upper phase was extracted using 200 µL of PCA twice. RNA in the aqueous phase was then precipitated with 100% ethanol. After washing the RNA pellet with 75% ethanol, the pellet was air dried and dissolved in appropriate volume of double distilled H2O.
8
Fecal Microbiome DNA Extraction Protocol
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Human fecal samples were collected in sterile transparent tubes with a screw cap (L × Ø: 76 × 20 mm) (SARSTEDT) containing RNAlater® (Ambion). After samples were weighed, RNAlater® was added to generate tenfold dilutions of homogenates. Homogenates (200 µl) were washed twice with 1 ml of phosphate-buffered saline and stored at − 20 °C until use. Bacterial DNA was extracted according to a previously described method [20 (link)]. Briefly, to extract DNA, 300 µl of Tris-SDS solution, 0.3 g of glass beads (diameter, 0.1 mm, BioSpec Products), and 500 µl of Tris–EDTA-saturated phenol were added to the suspension, and the mixture was vortexed vigorously using a FastPrep-24 (M.P. Biomedicals) at a power level of 5.0 for 30 s for bacterial DNA and 60 s for fungal DNA. After centrifugation at 20,000×g for 5 min, the supernatant (400 µl) was collected. Subsequently, phenol–chloroform extraction was performed, and the supernatant of 250 µl was subjected to isopropanol precipitation. Finally, DNA was suspended in 200 µl of TE buffer and stored at − 20 °C.
9
Yeast Strains and Lipid Reagents for Biochemical Studies
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Yeast strains and plasmids used in this study are listed in Supplemental Tables S3 and S4, respectively. Lipids were purchased from Avanti Polar Lipids, except for all diC16 phosphatidylinositols/phosphatidylinositol phosphates (Echelon Biosciences). All other chemicals were purchased from Sigma-Aldrich, except for glass beads (0.5 mm; BioSpec Products); Pierce 660 nm Protein Assay (Thermo Scientific); n-dodecyl β-d -maltoside (DDM) and octyl β-d -glucopyranoside (OG) (Glycon Biochem); ATP (PanReac AppliChem); BSA-free RGS-His Antibody (Qiagen); agar, yeast extract, and peptone (Becton, Dickinson and Co.); and γ-32P-ATP, 32P-orthophosphate, and myo-inositol-[3H] (Perkin-Elmer).
Yeast cells were grown in YPD (1% wt/vol yeast extract, 2% wt/vol bactopeptone, 2% wt/vol glucose) or YPG (1% wt/vol yeast extract, 2% wt/vol bactopeptone, 2% wt/vol galactose) medium. Selection was performed on a synthetic defined minimal medium (0.7% wt/vol yeast nitrogen base, 0.02 mg/ml amino acid, 50 mM succinic acid-Tris, pH 5.5) containing the appropriate dropout nutritional supplements (Sigma) and 2% wt/vol glucose (SD) or galactose (SG). For plates, 2% wt/vol agar was added. Unless otherwise stated, yeast was transformed using the Li-Ac method (Gietz et al., 1995 (link)).
Yeast cells were grown in YPD (1% wt/vol yeast extract, 2% wt/vol bactopeptone, 2% wt/vol glucose) or YPG (1% wt/vol yeast extract, 2% wt/vol bactopeptone, 2% wt/vol galactose) medium. Selection was performed on a synthetic defined minimal medium (0.7% wt/vol yeast nitrogen base, 0.02 mg/ml amino acid, 50 mM succinic acid-Tris, pH 5.5) containing the appropriate dropout nutritional supplements (Sigma) and 2% wt/vol glucose (SD) or galactose (SG). For plates, 2% wt/vol agar was added. Unless otherwise stated, yeast was transformed using the Li-Ac method (Gietz et al., 1995 (link)).
10
Cecal Short-Chain Fatty Acid Profiling
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Ceca of mice were harvested, frozen immediately in liquid nitrogen, and stored at −80°C until further processing. One milligram of cecal content was mixed with 10 μl 70% ethanol solution, and then homogenized with appropriate amounts of glass beads (1.0 mm in diameter; Biospec Products) by vortexing at 3,000 rpm for 10 min. Homogenized samples were centrifuged at 14,000 × g for 10 min, and the supernatants were collected and processed for fatty acid derivatization according to the method described previously (44 (link)). The derivatized supernatants were filtered using a 0.22-μm polycarbonate syringe filter (Millipore, St. Charles, MO, USA). Analysis of SCFAs was performed using high-performance liquid chromatography (HPLC) (HITACHI, Tokyo, Japan). SCFAs were separated using a C18 HTec column (NUCLEODUR, Macherey-Nagel, Düren, Germany) with a 40°C column temperature, flow rate at 1 ml/min, and detection wavelength set to 400 nm.
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