Exponential cultures of S. pombe growing at 30ºC in 400 ml YES with an OD600 0.8 were fixed by 1% formaldehyde at room temperature for 5 min followed by the addition of glycine to 0.125 M final concentration. The fixed cells were washed with ice-cold PBS, resuspended in cell lysis buffer [HEPES 50 mM (pH 7.6), EDTA 1 mM (pH 8.0), NaCl 150 mM, Triton X-100 1%, Na-Doc- 0.1% and 1X protease inhibitor (EDTA-free protease inhibitors cocktail tablet, Roche], and lysed by acid washed glass beads. Chromatin extracted from cell lysates was fragmented by 8 sonicating cycles of 5 min with 30 s ON/30s OFF at HIGH setting (Bioruptor® Plus). Immunoprecipitations were performed using Protein G Dynabeads (Life Technologies) coated with 10 μg monoclonal anti-Flag M2 antibody (Sigma). Both IP and Input DNA were purified using MinElute PCR Purification Kit (QIAGEN). 10 ng of DNA was used for DNA library construction with the NEBNext Ultra II DNA Library Prep Kit (New England Biolab E7645L), indexed using NEBnext Multiplex Oligos for Illumina Dual Index Primers (New England Biolabs E7600S) and sequenced simultaneously using a Illumina HiSeq4000 System.
Edta free protease inhibitors cocktail tablet
Manufactured by Roche
EDTA-free protease inhibitor cocktail tablet is a laboratory reagent used to prevent the degradation of proteins by proteolytic enzymes during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000 system, by Illumina (2 mentions)
Nebnext multiplex oligos for dual index primers, by Illumina (2 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
2 protocols using edta free protease inhibitors cocktail tablet
1
Chromatin Immunoprecipitation in Fission Yeast
2
Chromatin Immunoprecipitation in S. pombe
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Exponential cultures of S. pombe growing at 30ºC in 400 ml YES with an OD600 of 0.8 were fixed by 1% formaldehyde at room temperature for 5 min followed by the addition of 0.125 M glycine. The fixed cells were washed with ice-cold PBS, resuspended in cell lysis buffer [HEPES-50 mM (pH 7.6), EDTA-1 mM (pH 8.0), NaCl-150 mM, Triton X-100-1%, Na-Doc-0.1% and 1X protease inhibitor (EDTA-free protease inhibitors cocktail tablet, Roche], and lysed by acid washed glass beads. Chromatin extracted from cell lysates was fragmented by 8 sonicating cycles of 5 min with 30s ON/30s OFF at HIGH setting (Bioruptor® Plus). Immunoprecipitations were performed using Protein G Dynabeads (Life Technologies) coated with 10 µg monoclonal anti-FLAG M2 antibody (Sigma). Both IP and Input DNA were purified using MinElute PCR Purification Kit (QIAGEN). 10 ng of DNA was used for DNA library construction with the NEBNext Ultra II DNA Library Prep Kit (New England Biolab E7645L), indexed using NEBnext Multiplex Oligos for Illumina Dual Index Primers (New England Biolabs E7600S) and sequenced simultaneously using a Illumina HiSeq4000 System.
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