Each frozen specimen was transferred to a 1.5 mL tube supplied for use with a PowerMasher 2 (Nippi, Tokyo, Japan) and mixed with phase transfer surfactant (PTS) buffer supplemented with cOmplete™ protease inhibitor and PhosStop™ phosphatase inhibitor57 (link). Each specimen was homogenized for 30 s, and subjected to boiling at 95 °C for 5 min. The cell lysates were further sonicated twice (15 min per set) with a Bioruptor sonicator (Cosmo Bio, Tokyo, Japan). The cell lysates were centrifuged at 20,000 rcf at 4 °C for 3 min and the supernatants of the corresponding three specimens were transferred to a new tube. After centrifugation, the protein concentration was measured with a detergent compatible (DC) protein assay kit.
Bioruptor sonicator
Manufactured by Cosmo Bio
Sourced in Japan
The Bioruptor sonicator is a laboratory instrument used for the fragmentation of biological samples, such as DNA, RNA, and proteins, through the application of ultrasonic energy. It is designed to provide efficient and reproducible sample disruption for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Power masher 2, by Nippi (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Protein g sepharose, by Cytiva (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
Gotaq qpcr master mix, by Promega (2 mentions)
Cp100wx, by Hitachi (2 mentions)
Glomax multi detection system, by Promega (1 mentions)
Nano glo luciferase assay kit, by Promega (1 mentions)
Optiplate 96, by PerkinElmer (1 mentions)
Protein a agarose salmon sperm dna, by Merck Group (1 mentions)
7900ht fast real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Control rabbit igg, by Jackson ImmunoResearch (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Pierce microplate bca protein assay kit reducing agent compatible, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Lys c, by Fujifilm (1 mentions)
18 protocols using bioruptor sonicator
1
Protein Extraction from Frozen Specimens
2
Quantifying Transient Gene Expression in Plant Protoplasts
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107 protoplasts were co-transformed with 3 µg of the NanoLuc vector and 3 µg of the GFP vector pCop007. Transformed protoplasts were incubated at 28 °C for 16–24 hrs. After incubation, protoplasts were centrifuged for 5 min at 600 g. Pellets were re-suspended in 100 µl of phosphate buffer (pH 5.5), mixed by pipetting, and disrupted by 10 time cycles of the 30 sec-sonication with the Bioruptor sonicator (CosmoBio) with HIGH setting/the 30 sec-cooling down on ice. The cell lysates were transferred to OptiPlate-96 (PerkinElmer), and the Luc activity quantified with a Nano-Glo Luciferase assay kit (Promega) following the manufacturer’s protocol. Luminescence was measured with a luminometer GloMax®-Multi Detection System (Promega). Normalized luminescence of each sample was calculated by dividing the luminescence by the transformation rate as following.
3
ChIP-seq of H3K4me3 Enrichment
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ChIP using antibodies against trimethyl H3K4me3 (Millipore) was carried out according to a previously described method [49 (link)]. Briefly, cultured cells were cross-linked in 1% formaldehyde for 10 min at 37 °C. After the addition of 1/10 volume of 1.25 M glycine and incubation for 5 min, fixed cells were washed twice with cold PBS buffer. Soluble chromatin was prepared by sonication (Bioruptor sonicator; Cosmo Bio) to an average DNA size of 500 bp in sonication buffer and immunoprecipitated in IP buffer (20 mM Tris–HCl, pH 8.0, 600 mM NaCl, 1 mM EDTA, 0.05% SDS, 1.0% Triton X-100, 20% glycerol, 1.5 μM aprotinin, 10 μM leupeptin, 1 mM DTT and 40 μM MG132). Protein G sepharose (Amersham, USA) blocked with BSA was added and the antibody–chromatin complex recovered by centrifugation. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real-time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). Primers for alphoidtetO repeat (tetO) as well as for a control 5S ribosomal DNA, are listed in Supplementary Table S1. At least three independent ChIP experiments were performed to estimate the level of enrichment.
4
ChIP-seq Protocol for Chromatin Immunoprecipitation
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ChIP was carried out according to a previously described method (31 (link)). Cultured cells were cross-linked in 1% formaldehyde for 10 min at 37°C. After addition of 1/10 volume of 1.25 M glycine and incubation for 5 min, fixed cells were washed twice with cold phosphate buffered saline (PBS) buffer. Soluble chromatin was prepared by sonication in water bath-based sonicator (Bioruptor sonicator; Cosmo Bio) to an average DNA size of 500 bp and immnnoprecipitated in IP buffer (0.01% sodium dodecyl sulphate (SDS), 1.1% Triton X- 100, 1.2 mM ethylenediaminetetraacetic acid, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). Protein A agarose/salmon Sperm DNA (Millipore, #16–157) was added, and the antibody-chromatin complex was recovered by centrifugation. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a 7900HT Fast real-time PCR detection system (Applied Biosystems) and SYBR Green PCR Master Mix (Applied Biosystems, #4309155). Primers for HORs regions of chromosome 21 and chromosome X, tetO-2 mer of the alphoidtetO DNA array, HORs D5Z1 and D5Z2 regions of chromosome 5 were described previously (30 (link),32 (link)) (see also Supplementary Table S1).
5
ChIP-seq of Histone Modifications
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ChIP with antibodies against trimethyl H3K4me3 (Upstate) and CENP-A was carried out according to a previously described method (40 (link)). Briefly, cultured cells were cross-linked in 1% formaldehyde for 10 min at 37°C. After addition of 1/10 volume of 1.25 M glycine and incubation for 5 min, fixed cells were washed twice with cold PBS buffer. Soluble chromatin was prepared by sonication (Bioruptor sonicator; Cosmo Bio) to an average DNA size of 500 bp in sonication buffer and immunoprecipitated in IP buffer (20 mM Tris-HCl, pH 8.0, 600 mM NaCl, 1 mM EDTA, 0.05% SDS, 1.0% Triton X-100, 20% glycerol, 1.5 μM aprotinin, 10 μM leupeptin, 1 mM DTT and 40 μM MG132). Protein G sepharose (Amersham, USA) blocked with BSA was added, and the antibody–chromatin complex was recovered by centrifugation. The recovery ratio of the immunoprecipitated DNA relative to input DNA was measured by real-time PCR using a CFX96 real time PCR detection system (Bio-Rad) and iQ SYBR Green Supermix (Bio-Rad). Primers for alphoidtetO repeat (tetO) as well as for control loci, 5S ribosomal DNA, are listed in Supplementary Table S1. At least three independent ChIP experiments were performed to estimate the level of enrichment.
6
Tissue Lysis and Protein Extraction
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Each frozen sample was transferred to a 1.5-mL tube and mixed with phase transfer surfactant (PTS) buffer (50 mM ammonium bicarbonate, 12 mM sodium deoxycholate, 12 mM sodium lauroyl sarcosinate) supplemented with cOmplete™ protease inhibitor and PhosStop™ phosphatase inhibitor 20 (link). Each biopsy was homogenized in a PowerMasher 2 (Nippi, Tokyo, Japan) for 30 seconds and subsequently boiled at 95°C for 5 min. The lysates were further sonicated three times (15 min per cycle) with a Bioruptor sonicator (Cosmo Bio, Tokyo, Japan). After sonication, the protein concentration was measured with DC protein assay kit.
7
FAIRE Analysis of Chromatin Accessibility
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Eighteen hours prior to analysis, J-Lat A2 and 11.1 cells were treated with BAFi's where indicated. J-Lat cells were fixed for 30 min by adding formaldehyde to a final concentration of 1% at room temperature. Twenty million cells were used per FAIRE experiment. The reaction was quenched with 125 mM glycine. Cross-linked cells were washed with PBS followed by washes with buffer B (0.25% Triton-X 100, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, pH 7.6) and buffer C (150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, pH 7.6). For sonication, cells were re-suspended in ChIP incubation buffer (1% SDS, 1% Triton-X 100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, pH 7.6) and chromatin was sheared by sonication to an apparent length of ~ 200–400 bp (corresponding to ~ 100–200 bp of free DNA) using a BioRuptor sonicator (Cosmo Bio Co., Ltd) with 20 times 30-s pulses at maximum setting at 4 °C. Sonicated chromatin was twice phenol:chloroform:isoamyl alcohol (24:24:1) extracted, washed with chloroform:isoamylalcohol (24:1) and ethanol precipitated. Isolated DNA was subjected to Sybergreen qPCR cycles with specific primers (sequences provided in Table. 1 ) with a CFX Connect Real-Time PCR Detection System (BioRad) and GoTaq qPCR Mastermix (Promega), using following thermal program starting with 3 min at 95 °C, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s.
8
Quantifying Intracellular and Extracellular EBV DNA
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Following lysis and sonication using a Bioruptor sonicator (Cosmobio, Tokyo, Japan; 5 min, 30-s on/off pulses), genomic DNA (gDNA) was extracted from 125 μM cordycepin-treated SNU719 cells. The resultant gDNA (50 ng) was subjected to qPCR analysis, and the relative amount of EBV gDNA was determined using the internal control GAPDH. Intracellular EBV copy number was calculated as the relative amount of EBV gDNA in the total gDNA. To determine the relative extracellular EBV copy number, 20-ml culture medium samples were collected from SNU719 cells treated with cordycepin. The culture medium samples were filtered through a 0.45-nm syringe filter, loaded onto a 20% sucrose cushion in phosphate-buffered saline (PBS) solution, and subjected to ultracentrifugation (CP100WX, Hitachi) at 27,000 rpm for 90 min. The virus pellet was lysed in 100 μl of FA lysis buffer [EDTA (1 mM, pH 8.0), HEPES-KOH (50 mM, pH 7.5), and NaCl (140 mM)], sonicated using a Bioruptor sonicator for 5 min (30-s on/off pulses), and DNA was extracted. Finally, viral DNA was resuspended in 100 μl of RNase-free water and qPCR analysis was used to quantify viral DNAs by using primer sets specific for EBV EBNA1.
9
ChIP Assay for GLI1 Binding
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ChIP assays were performed essentially as described previously.38 B16F10 cells were cross‐linked with formaldehyde, lysed, and sonicated using a Bioruptor sonicator (CosmoBio, Tokyo, Japan). The lysates were immunoprecipitated with the rabbit anti‐GLI1 H300 Ab (sc‐20687; Santa Cruz Biotechnology) or control rabbit IgG (011‐000‐003; Jackson ImmunoResearch Laboratory, West Grove, PA, USA), and the precipitated DNA was subjected to quantitative PCR (qPCR). Primers used for the qPCR are listed in Table S3 .
10
Urban Particulate Matter Effects on Cells
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Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). PD98059, SP600125, and SB203580 were purchased from Calbiochem (La Jolla, CA, USA). Ammonium pyrrolidinedithiocarbamate (PDTC), forskolin (Fk), capsaicin, lipopolysaccharide (LPS), and Phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The protease inhibitor cocktail was purchased from Roche (Indianapolis, IN, USA). Urban particulate matter (UPM) (the standard reference material, 1648a) was supplied by National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). The composition of UPM was shown in Table 1 . UPM was suspended in deionized water at a concentration of 50 mg/mL and stored at −20 °C until used. UPM stock solution was sonicated for 5 min using Bioruptor sonicator (Cosmobio, Tokyo, Japan) immediately before treatment. Working solution of UPM was prepared in 500 μL DMEM and added to the cultured cells.
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