ChIP was carried out in WT total bone marrow TER119+ cells as previously described [17 (link)]. Briefly, cells were cross-linked in 0.4% formaldehyde in PBS, and lysed (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% NP40). Lysate was sonicated for 30 cycles of 30 s on/30 s off at 4C° using a Bioruptor Standard sonication device (Diagenode). The cell lysate was then diluted in ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA 150 mM NaCl, 1% Triton, 0.01% SDS) and incubated at 4C° overnight with anti-FOXO3a antibody (Millipore #07–702) and Magna ChIPTM Protein A+G magnetic beads (Millipore #16–663). Beads were then washed (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 50 mM NaCl, 1% Triton, 0.1% SDS) and recovered. The antibody-protein-DNA complexes were reverse cross-linked for DNA isolation and quantitative PCR (qPCR) analysis. Foxo3-/- TER119+ cells were used as negative controls. Putative binding sites were located using MatInspector from Genomatix (http://www.genomatix.de/ ). Primer specific sequences are listed in S11 Table . See S12 Table for all antibodies.
Bioruptor standard sonication device
Manufactured by Diagenode
The Bioruptor Standard is a sonication device used for the mechanical disruption of biological samples. It utilizes ultrasonic waves to fragment DNA, RNA, and proteins for various applications in molecular biology and genomics.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase inhibitor cocktail, by Merck Group (2 mentions)
Magna chiptm protein a g magnetic beads, by Merck Group (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Ion personal genome machine system, by Thermo Fisher Scientific (1 mentions)
Ab library builder system, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 instrument 2, by Roche (1 mentions)
Complete mini protease inhibitor cocktail tablet, by Roche (1 mentions)
7 protocols using bioruptor standard sonication device
1
ChIP-qPCR Analysis of FOXO3a Binding in Bone Marrow
2
Histone Modification Analysis via Cell Lysis and Chromatin Extraction
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Whole cell extracts were obtained from cell lines in log phase of growth or from purified mouse B cells using NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris-HCl pH 8.0) supplemented with proteinase inhibitor cocktail (Sigma-Aldrich) and 0.5 mM phenylmethanesulfonyl fluoride (PMSF) (Roche), according to previously described protocols61 (link). For the analysis of histone modifications, the chromatin pellet was subsequently resuspended in RIPA-coIP buffer (Tris-HCl pH 8.0 50mM, NaCl 200mM, Glycerol 5%, MgCl2 2.5mM, Sodium deoxycholate 0.05%, SDS 0.05%) and mobilized by sonication in a Bioruptor Standard sonication device (Diagenode). Extracts were cleared by centrifugation at 12,000 rpm for 10 minutes.
3
Histone Modification Analysis via Cell Lysis and Chromatin Extraction
4
ChIP-Seq Protocol for HeLa Cells
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For each of the three replicates, 20 × 106 HeLa cells were fixed with 1% formaldehyde for 15 min to cross-link protein:DNA complexes, followed by a quench with 125 mM glycine. Cells were washed with cold 0.01 M PBS (pH 7.4) and lysed for 10 min at 40°C in cell lysis buffer (50 mM HEPES (pH 8.0), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100). Cellular nuclei were then washed (10 mM Tris–HCl (pH 8.0), 200 mM EDTA), centrifuged and re-suspended in nuclear lysis buffer (50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, 1% SDS). A Diagenode Bioruptor Standard sonication device (run at max amplitude for 5 × 15 min in ice water) was used to shear the cross-linked DNA to 100–400 bp fragments. Proteinase K digestion was performed overnight at 55°C. DNA was purified with the MinElute PCR Purification Kit (Qiagen). DNA quantification, library construction and sequencing were all performed by the High-Throughput Genomics Core within the University of Utah Huntsman Cancer Institute. The input DNA libraries were sequenced using the Illumina HiSeq 2000 platform.
5
Targeted DNA Sequencing Protocol
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The amplicons of each patient were pooled and later fragmented in 200 pb using a Bioruptor ® standard sonication device (Diagenode). Libraries were built using the automated AB Library Builder System (Thermo Fisher Scientific), using Ion ™ Plus Library Kit. To normalizing the number of molecules required for emulsion PCR (ePCR), a quantitation step was performed in LightCycler® 480 Instrument II (Roche Life Science). The ePCR was performed in an automated Ion OneTouch 2 platform (Thermo Fisher Scientific) in accordance with manufacturer protocols. Finally, the mix was loaded on an Ion 318™ Chip Kit-Ion Torrent ™ (Thermo Fisher Scientific) and sequencing reactions were performed on an Ion Personal Genome Machine™ (PGM™) System using Ion PGM™ HI-Q™ View Sequencing (Thermo Fisher Scientific) kit.
6
Histone Modification Analysis in B Cells and Tumor Biopsies
7
Bacterial Protein Extraction and Quantification
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Four-day old colonies of monocultures of the selected bacterial strains were suspended in sterile phosphate buffered saline (PBS) with protease inhibitors (Complete Mini Protease Inhibitor Cocktail Tablets, Roche Diagnostics, 1 tablet for 7 ml PBS). After washing and centrifugation cycles (3 × 5 min., 14489 g, 4 °C) the bacterial pellets were resuspended in lysis buffer containing non-denaturing detergent (Noninet P-40, Sigma-Aldrich, Inc.) and sonicated on ice for 15 min. (Bioruptor® Standard sonication device, Diagenode s.a.). Protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific, Pierce Protein Biology Products).
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