Quantitative Chromatin ImmunoPrecipitation (qChIP) was performed as described (Pidoux et al., 2004 (link)) with the following modifications: 5 ml of an overnight culture grown in YPAD with extra uridine (0.08 mg/ml), diluted into fresh YPAD with extra uridine (0.08 mg/ml) and grown until OD600 nm of 1.4. Cells (50 ml/sample) were fixed with 1% Paraformaldehyde (Sigma) for 15 min at room temperature. Cells were lysed using acid-washed glass beads (Sigma) and a Disruptor genieTM (Scientific Industries) for 30 min at 4°C. Chromatin was sheared to 500–1000 bp using a Bioruptor (Diagenode) for a total of 20 min (30 s ON and OFF cycle) at 4°C. Immunoprecipitation was performed overnight at 4°C using 2 μL of antibody anti-H3K4me2 (Active Motif- Cat Number: 39141), anti-H3K9ac (Active Motif- Cat Number: 39137), and anti-H4K16ac (Active Motif- Cat Number: 39167) and 25 μl of Protein G magnetic beads (Dynal – InVitrogen). DNA was eluted with a 10% slurry of Chelex 100-resin (Bio-Rad) using the manufacturer’s instructions.
Disruptor genie
Manufactured by Scientific Industries
Sourced in United States
The Disruptor Genie is a laboratory equipment device designed for the disruption of biological samples. It utilizes a high-speed mechanical agitation system to effectively disrupt cells, tissues, or other materials for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Zirconia silica bead, by Biospec (5 mentions)
Acid washed glass beads, by Merck Group (4 mentions)
Allprep dna rna mini kit, by Qiagen (4 mentions)
Br717805 1ea, by Merck Group (3 mentions)
Rlt lysis buffer, by Qiagen (3 mentions)
Pepstatin a, by Thermo Fisher Scientific (3 mentions)
Chelex 100 resin, by Bio-Rad (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Bioruptor, by Diagenode (2 mentions)
Gene prep star pi 480, by Kurabo (2 mentions)
Bond breaker tcep solution, by Thermo Fisher Scientific (2 mentions)
Glass bead, by Merck Group (2 mentions)
Zetasizer nano, by Malvern Panalytical (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rlt buffer, by Qiagen (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Anti h3k9ac, by Active Motif (1 mentions)
Anti h4k16ac, by Active Motif (1 mentions)
60 protocols using disruptor genie
1
Quantitative Chromatin Immunoprecipitation Protocol
2
Chromatin Immunoprecipitation with Histone Modifications
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qChIP was performed as described33 (link) with the following modifications: 5 ml of an overnight culture grown in YPAD with extra uridine (0.08 mg/ml), diluted into fresh YPAD with extra uridine (0.08 mg/ml) and grown until OD600 nm of 1.4. Cells (50 ml/sample) were fixed with 1% Paraformaldehyde (Sigma) for 15 min at room temperature. Cells were lysed using acid-washed glass beads (Sigma) and a Disruptor genieTM (Scientific Industries) for 30 min at 4 °C. Chromatin was sheared to 500–1000 bp using a Bioruptor (Diagenode) for a total of 20 min (30 s ON and OFF cycle) at 4 °C. Immunoprecipitation was performed overnight at 4 °C using 2 μL of antibody anti-H3K4me2 (Active Motif- Cat Number: 39141), anti-H3K9ac (Active Motif- Cat Number: 39137), and anti-H4K16ac (Active Motif- Cat Number: 39167) and 25 μl of Protein G magnetic beads (Dynal - InVitrogen). DNA was eluted with a 10% slurry of Chelex 100-resin (Bio-Rad) using the manufacturer’s instructions.
3
RNA Extraction from Bacterial Cultures
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Quantities of 5 U (OD600) of culture samples were mixed with twice the volume of bacterial RNAprotect (Qiagen, Valencia, CA) and centrifuged for 10 min at 5,000 rpm. The supernatants from these samples were discarded and cell pellets were mixed with 1 mL of TRIzol (Invitrogen, Carlsbad, CA), transferred to lysing matrix B tubes (MP Biomedicals, Irvine, CA), and subjected to lysis using Disruptor Genie (Scientific Industries, Bohemia, NY) for 4 min at 3,000 rpm. The cell lysate was processed using an RNeasy kit (Qiagen) per the manufacturer’s instructions. The concentration and purity of isolated RNA were determined using a NanoDrop 2000 UV-visible (UV-Vis) spectrophotometer (Thermo Fisher Scientific, Waltham, MA).
4
Automated Bacterial DNA Extraction from Diverse Samples
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Bacterial DNA extraction from vaginal, rectal, and oral samples was carried out by BIKEN Biomics Inc. (Osaka, Japan) using an automated DNA extraction machine (GENE PREP STAR PI-480, Kurabo Industries Ltd., Osaka, Japan) and NR-201 DNA extraction kit (Kurabo Industries Ltd., Osaka, Japan). DNA was extracted according to manufacturer’s protocol. In brief, 0.5 g of glass beads (0.1 mm diameter, IEDA Trading Corp., Tokyo, Japan) and 300 μL of No. 10 solution (NR-10025) were added to each stool suspensions (200 μL each), following which the mixture was agitated using DISRUPTOR-GENIE (Scientific Industries Inc., New York, USA) at 3000 rpm for 90 s. After centrifugation at 9700× g for 5 min, the supernatant of stool samples was collected and transferred to the machine fitting strips of eight sample tubes. Next, 150 μL of No. 2 solution (NR-2025) supplemented with proteinase K (final concentration 0.4 mg/mL, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 150 μL of No. 10 solution (NR-10025) were added to the sample solution and the tubes were subjected to the automated DNA extraction machine, GENE PREP STAR PI-480. The concentration of extracted DNA was determined using Qubit assays (Thermo Fisher Scientific Inc., Delaware, USA). Extracted DNA samples were stored at −30 °C until use.
5
Preparation and Milling of DCF Nanosuspension
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DCF was prepared from a sodium salt solution following a published procedure by Pireddu et al., using diluted hydrochloric acid [12 (link)]. The obtained solid white precipitate of DCF was filtered, carefully washed with bidistilled water and dried. To prepare the nanosuspension, the drug was firstly added to an aqueous solution of P188 and homogenized by means of a rotor-stator homogenizer (Ultra Turrax T25 basic—IKA, Staufen, Germany) for 5 min at 6500 rpm. Secondly, the obtained suspension was transferred in conical tubes, and 0.1–0.2 mm yttrium-stabilized zirconia–silica beads were added to perform the milling. The tubes were then oscillated at 3000 rpm for 60 min by means of a bead-milling cell disruptor device (Disruptor Genie®, Scientific Industries, Bohemia, NY, USA). Finally, sieving was carried out to separate the milling beads from the formulation. The resulting nanosuspension had a concentration of 10 mg/mL DCF and 5 mg/mL P188 (Table 1 ).
6
Isolation and Characterization of E. coli Cellular Components
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The cells and spent medium from a 10 ml E. coli pre-culture were separated by centrifugation at 1,500 g for 20 min at 4 °C. The supernatant was filter sterilized (0.2-µm cellulose membrane) and used for further analysis (“spent medium”). The collected cells were washed 3 times with phosphate-buffered saline (PBS, pH 7.0), re-suspended in 1.5 ml of PBS, and disrupted with sterile glass beads (diameter, 0.1 mm) using a cell disruptor (Disruptor Genie, Scientific Industries, NY, USA): a 2-ml tube containing 1.4 g beads and the cell suspension was mixed for 20 min with intervals to cool the tube in an ice-water bath. Following disruption, the volume of the mixture was raised to a total volume of 10 ml using PBS and centrifuged at 10,000 g for 20 min at 4 °C. The resulting supernatant was filtrated (0.2-µm cellulose membrane) and used as a cell-free extract for further analysis.
7
Isolating and Excysting Eimeria Oocysts
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Eimeria spp. oocysts were collected from fecal samples of nonvaccinated animals showing coccidiosis symptoms. The samples were processed as indicated in Guidelines on techniques in coccidiosis research (Shirley, 1995 ) with some changes. Oocysts were resuspended in potassium dichromate 2% (Cat.#P5271, Sigma-Aldrich, St. Louis, MO) to allow sporulation. The oocyst samples were cleaned with Dulbecco's modified phosphate buffered saline (DPBS , Cat.#D8537, Sigma-Aldrich) from potassium dichromate, and then, they were resuspended in lysis buffer T1 (Cat.# 740952.240 C, MACHEREY-NAGEL Inc., Bethlehem, PA) and stored at −80°C until qPCR analysis was performed as described below. After sporulation, the oocysts were washed and resuspended in sodium hypochlorite for sterilization, and then, they were washed and lysed with glass beads (0.5 mm) for 1 min with Disruptor Genie (Cat.# SI-D258, Scientific Industries, Bohemia, NY) to obtain sporocysts. Those were washed and resuspended in excystation medium, containing 2.5 g/L trypsin (Cat.#T4049, Sigma-Aldrich), 5 g/L bile salts (Cat.#B3301, Sigma-Aldrich), 2 g/L pancreatin (Cat.#P1750, Sigma-Aldrich), and 2 g/L MgCl2 (Cat.# 459337, Carlo Erba Reagents, Milan, Italy). The suspension was incubated for 90 min at 39°C. Afterward, the obtained sporozoites were washed and resuspended in cell medium to initiate the invasion assay.
8
Sperm RNA Extraction Protocols
9
Sperm DNA Extraction from Ejaculates
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Extraction of sperm cell DNA from fresh ejaculates was performed as previously described40 (link). In short, sperm cells were isolated by centrifugation of the fresh (up to 2 days) ejaculate over an isotonic solution (90%) (Sage/Origio, ART-2100; Sage/Origio, ART-1006) using up to 2 mL of the sample. Following a washing step, quantity and quality were assessed using a cell counting chamber (Sigma-Aldrich, BR717805–1EA). Cells were pelleted and lysis was performed by addition of RLT lysis buffer (Qiagen, 79216), Bond-Breaker TCEP solution (Pierce, 77720), and 0.2 mm stainless steel beads (Next Advance, SSB02) on a Disruptor Genie (Scientific Industries, SI-238I). The lysate was processed using reagents and columns from an AllPrep DNA/RNA Mini Kit (Qiagen, 80204). Concentration of the final eluate was assessed employing standard methods. Concentrations ranged from ~0.5–300 ng/μl.
10
Efficient Sperm DNA Extraction Protocol
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