Freeze fracture EM analysis was performed as described previously28 (link). Briefly, yeast cells sandwiched between a 20-μm–thick copper foil and a flat aluminum disc (Engineering Office M. Wohlwend, Sennwald, Switzerland) were quick-frozen by high-pressure freezing using an HPM 010 high-pressure freezing machine according to the manufacturer’s instructions (Leica Microsystems, Wetzlar, Germany). Frozen specimens were transferred to the cold stage of a Balzers BAF 400 apparatus and fractured at − 120° under a vacuum of ~ 1 × 10−6 mbar. Freeze-fractured samples were subjected to a three step electron-beam evaporation: C (6 nm) at 90° Pt/C (2 nm) at 45°, and C (10 nm) as previously described28 (link). Thawed replicas were treated with 2.5% SDS in 0.1 M Tris–HCl (pH 8.0) at 60 °C overnight, with 0.1% Westase (Takara Bio, Kusatsu, Japan) in McIlvain buffer (37 mM citrate, 126 mM disodium hydrogen phosphate, pH 6.0) containing 10 mM EDTA, 30% fetal calf serum, and a protease inhibitor cocktail for 90 min at 30 °C, with 2.5% SDS again in 0.1 M Tris–HCl (pH 8.0) at 60 °C overnight. Replicas were observed and photographed with a JEOL JEM-1011 EM (Tokyo, Japan) using a CCD camera (Gatan, Pleasanton, CA, USA).
Westase
Manufactured by Takara Bio
Sourced in Japan
Westase is a laboratory equipment product designed for DNA and RNA purification. It utilizes a proprietary technology to efficiently extract and purify nucleic acids from various biological samples. The core function of Westase is to provide a reliable and consistent method for the isolation and concentration of DNA and RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Genomic tip 20 g column, by Qiagen (3 mentions)
Nucleospin rna plant and fungi mini kit, by Macherey-Nagel (3 mentions)
Baf 400, by Oerlikon Balzers (2 mentions)
Hpm 010 high pressure freezing machine, by Leica (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Ccd camera, by Ametek (1 mentions)
Sepasol rna 1 super, by Nacalai Tesque (1 mentions)
Truseq rna sample prep kit, by Illumina (1 mentions)
Truseq dna sample preparation kit, by Illumina (1 mentions)
Genomic tip 100 g, by Qiagen (1 mentions)
Hexokinase, by Oriental Yeast (1 mentions)
Glucose 6 phosphate dehydrogenase, by Roche (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Zymolyase 100t, by Nacalai Tesque (1 mentions)
Bovine serum albumin (bsa), by Nacalai Tesque (1 mentions)
8 protocols using westase
1
Yeast Freeze Fracture Electron Microscopy
2
Genomic and Transcriptomic DNA/RNA Extraction
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For genomic DNA extraction, cultured cells were lysed with Westase (Ozeki, Japan) following the manufacturer’s instructions for Saccharomyces cerevisiae. Genomic DNA was extracted following Westase’s protocol provided by the distributor (Takara, Japan). Isolated DNA was purified using Genomic-tip 20/G columns (Qiagen, Netherlands). For RNA extraction, cells were twice disrupted using a vortex mixer with glass beads for 30 s. RNA was extracted using a NucleoSpin RNA Plant and Fungi Mini Kit (Macherey-Nagel, Germany) following the manufacturer’s instructions.
3
Genomic and Transcriptomic DNA/RNA Extraction
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For genomic DNA extraction, cultured cells were lysed with Westase (Ozeki, Japan) following the manufacturer’s instructions for Saccharomyces cerevisiae. Genomic DNA was extracted following Westase’s protocol provided by the distributor (Takara, Japan). Isolated DNA was purified using Genomic-tip 20/G columns (Qiagen, Netherlands). For RNA extraction, cells were twice disrupted using a vortex mixer with glass beads for 30 s. RNA was extracted using a NucleoSpin RNA Plant and Fungi Mini Kit (Macherey-Nagel, Germany) following the manufacturer’s instructions.
4
Yeast Genomic DNA and RNA Extraction
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Yeast-like fungi were cultivated in YPD medium (0.5% (w/v) yeast extract, 1% peptone, and 0.9% glucose) at 25 °C for 3 days. The cells were collected from the medium by centrifugation (for 5 min at 3,000 × g) and were suspended in cell-wall lysis buffer (0.1 M McILvaine buffer containing 270 U/ml Westase (Takara Bio) and 0.5 M sodium tartrate). After incubation at 30 °C for 1.5 h, genomic DNA was extracted using Genomic-tip 100/G (Qiagen) according to manufacturer’s instruction. One microgram of the DNA was used to construct standard 300 bp libraries using the TruSeq DNA Sample Preparation Kit with the standard protocol (Illumina). Libraries were sequenced on an Illumina HiSeq2000 following the manufacturer’s recommended protocol to produce 100-bp paired end reads (https://icom.illumina.com/ , last accessed April 4, 2014). RNA was extracted from yeast cells grown on YPD agar medium using Sepasol-RNA I Super (Nacalai Tesque) after cell disruption by tungsten beads (φ0.1 mm). RNA libraries were prepared with an Illumina TruSeq RNA Sample Prep Kit and sequenced (100-bp paired end) on an Illumina HiSeq2000 sequencer using the standard protocol (Illumina).
5
Quantification of β-glucan in Cells
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Small-scale β-glucans were measured according to Hirokawa et al. (2008) (link)’s method with a slight modification. Briefly, cells in 2 mL of cultures at 3 days after RNAi were harvested by centrifugation (18,500 × g) and then suspended in 300 μL of the McIlvain buffer (pH6.0). After sonication, methanol was added to the suspension, and the mixture was incubated at 4°C for more than 4 h. After centrifugation, the β-glucan-containing pellet was dried up and then solved in 25 μL of the McIlvain buffer. The β-glucan was converted to glucose by treatment using westase (TaKaRa Bio), and the total glucose content was determined using hexokinase (Oriental Yeast Co., Ltd, Japan) and glucose-6-phosphate dehydrogenase (Roche Diagnostics K.K., Germany), according to Bergmeyer et al. (1974) . westase is an enzyme cocktail, including β-1,6-glucanase and β-1,3-glucanase, for yeast protoplast preparation, and it is demonstrated to completely degrade the β-glucan of P. haptonemofera into glucose (Hirokawa et al., 2008 (link)).
6
Freeze Fracture Replica Preparation
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A copper EM grid (100 mesh) immersed with yeast or liposome pellets was sandwiched between a 20-µm-thick copper foil and a flat aluminum disc (242; Engineering Office M. Wohlwend) and frozen using an HPM 010 high-pressure freezing machine (Leica). For freeze fracture, the sandwiched sample was transferred to the stage of a Balzers BAF 400 and fractured at −115°C to −105°C under a vacuum of ∼10−6 mbar. Replicas were made by electron-beam evaporation in three steps: carbon (C; 5 nm in thickness) at an angle of 90° to the fractured surface, platinum-C (2 nm) at an angle of 45°, and C (10 nm) at an angle of 90°. The thickness of evaporation was adjusted by referring to a quartz crystal thickness monitor.
Replicas were treated sequentially with 2.5% SDS in 0.1 M Tris-HCl (pH 8.0) overnight at 60°C, with 0.1 mg/ml Zymolyase 100T (07665–55; Nacalai) in PBS containing 0.1% Triton X-100, 1% BSA (01281–26; Nacalai), and a protease inhibitor cocktail (25955–11; Nacalai) for 2 h at 37°C, and again with 2.5% SDS in PBS overnight at 60°C. In some experiments, the Zymolyase step was replaced with digestion with 0.5% Westase (9005; Takara Bio) in McIlvain citrate phosphate buffer (pH 6.0) containing 10 mM EDTA and 30% FCS (Tsuji et al., 2019 (link)). The replicas were stored in buffered 50% glycerol in PBS at −20°C until use.
Replicas were treated sequentially with 2.5% SDS in 0.1 M Tris-HCl (pH 8.0) overnight at 60°C, with 0.1 mg/ml Zymolyase 100T (07665–55; Nacalai) in PBS containing 0.1% Triton X-100, 1% BSA (01281–26; Nacalai), and a protease inhibitor cocktail (25955–11; Nacalai) for 2 h at 37°C, and again with 2.5% SDS in PBS overnight at 60°C. In some experiments, the Zymolyase step was replaced with digestion with 0.5% Westase (9005; Takara Bio) in McIlvain citrate phosphate buffer (pH 6.0) containing 10 mM EDTA and 30% FCS (Tsuji et al., 2019 (link)). The replicas were stored in buffered 50% glycerol in PBS at −20°C until use.
7
Genomic and Transcriptomic DNA/RNA Extraction
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For genomic DNA extraction, cultured cells were lysed with Westase (Ozeki, Japan) following the manufacturer’s instructions for Saccharomyces cerevisiae. Genomic DNA was extracted following Westase’s protocol provided by the distributor (Takara, Japan). Isolated DNA was purified using Genomic-tip 20/G columns (Qiagen, Netherlands). For RNA extraction, cells were twice disrupted using a vortex mixer with glass beads for 30 s. RNA was extracted using a NucleoSpin RNA Plant and Fungi Mini Kit (Macherey-Nagel, Germany) following the manufacturer’s instructions.
8
Genomic DNA Extraction from N. liquefaciens
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N. liquefaciens N6 cells cultured in 5 ml of YPD were collected (3,500 × g, 5 min) and washed in protoplast buffer which is Mcllvain buffer [pH 6.0] (0.1 M citric acid solution and 0.2 M disodium hydrogenphosphate at the ratio of 36.8 : 63.2 [v/v] ) containing 0.3 M sodium tartrate. The cell pellet was resuspended in 0.5 ml westase solution (0.5% [w/v] of westase [Takara Bio, Japan] in protoplast buffer) and incubated for 1 h at 30°C. Cells were washed once in 1 ml of 50 mM Tris-HCl (pH 7.5), resuspended in 500 µl of TE 50:20 (50 mM Tris-HCl (pH 7.5) 20 mM EDTA), then mixed well with 50 µl of 10% SDS. After 30 min incubation at 65 °C, 200 µl of 5 M potassium acetate was added to the sample and stored on ice for 15 min. The sample was centrifugated at 15,000 rpm for 10 min to obtain the cell lysate, which was used for genomic DNA preparation by essentially the same procedure as for S.
cerevisiae (Amberg et al. 2015) (link). Alternatively, genomic DNA was purified using Dr.
GenTLE TM High Recovery kit (Takara Bio, Japan) from approximately 1x10 8 cells cultured in YPD. ｓ
cerevisiae (Amberg et al. 2015) (link). Alternatively, genomic DNA was purified using Dr.
GenTLE TM High Recovery kit (Takara Bio, Japan) from approximately 1x10 8 cells cultured in YPD. ｓ
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