Chromatin immunoprecipitation (ChIP) was performed as previously described (35 (link), 74 (link)). All ChIP-seq assays were performed using single donors in triplicate. For quantitative ChIP-seq experiments, 4 × 106 SF9 cells were spiked into the pool. The cells were treated with formaldehyde to cross-link, and chromatin was fragmented to 200 to 300 bp, using a Biorupter Pico sonicator (Diagenode). Each lysate was immunoprecipitated with 10 μg of anti-H3K4me3 (Merck Millipore) and anti-H3K27me3 (Merck Millipore) antibodies. The chromatin immunoprecipitation (ChIP) experiment was then performed for each antibody as described previously by Orlando et al. (74 (link)). Library preparation was performed using a NEBNext Ultra DNA sample preparation kit (NEB), according to the manufacturer’s recommendations. The samples were multiplexed, quantified using a High-sensitivity d1000 TapeStation (Agilent) or a Kapa library quantification kit (KAPA Biosystems), and then sequenced using a NextSeq 500 (Illumina) (paired-end, 2 × 41 bp). Sequencing depth was >20 million reads per sample.
Biorupter pico sonicator
Manufactured by Diagenode
Sourced in Belgium
The Biorupter Pico is a sonicator device designed for sample disruption and homogenization. It uses ultrasonic waves to break down samples, such as cells or tissues, into smaller fragments. The device is compact and easy to use, making it suitable for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext ultra dna sample preparation kit, by New England Biolabs (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Anti h3k27me3, by Merck Group (1 mentions)
Anti h3k4me3, by Merck Group (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Zymo spin chip kit, by Zymo Research (1 mentions)
Mrcf0r030, by Merck Group (1 mentions)
4 protocols using biorupter pico sonicator
1
Chromatin Immunoprecipitation (ChIP) Assay
2
ChIP-seq Analysis of BRD2 and BRD4 in NK Cells
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Chromatin immunoprecipitation (ChIP) was performed as previously described (20 (link)). All ChIPs were performed using single donors in triplicate. NK cells were cross-linked with formaldehyde, and chromatin was fragmented to 200 to 300 bp, using a Biorupter Pico sonicator (Diagenode). Immunoprecipitation was performed using 10 μg of anti-BRD2 (A302-583A, Bethyl Laboratories) or anti-BRD4 (A301-985A100, Bethyl Laboratories) antibodies. The ChIP experiment was then performed for each antibody as described previously by Orlando et al. (24 (link)). Next, library preparation was performed using a NEBNext Ultra DNA sample preparation kit (NEB), according to the manufacturer’s recommendations. The samples were then sequenced using a NextSeq 500 (Illumina) (single-end, 82 bp). Sequencing depth was >20 million reads per sample.
3
ChIP-Seq of ATF4, H3K4me3, and EWS-FLI1
4
Whole Proteome Peptide Preparation
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HEK293T cells transfected with indicated plasmids in biological triplicates were lysed with 8 M urea with 10 mM DTT and 0.1 M Tris (pH 8.5). Soluble cell lysates were collected after 10 cycles of sonication for 30 s followed by 30 s at 4 °C using a Biorupter Pico Sonicator (Diagenode, Liége, Belgium), followed by maximum speed centrifugation at room temperature for 15 min. Whole proteome peptides were prepared using the FASP protocol as described before [54] (link). In brief, 50 μg of soluble lysates were added onto 30 kDa cutoff filter (MRCF0R030, Sigma) and centrifuged at 11,000 r/min at 20 °C for 15 min. 50 mM IAA in urea buffer was used to alkylate proteins at 20 °C for 15 min. After 3 washes with urea lysis buffer and 3 washes with 50 mM ammonium biocarbonate (ABC; Catalog No. 09830, Sigma) buffer, 500 ng of trypsin in 50 μl of 50 mM ABC buffer was used to digest proteins in a wet chamber overnight at 37 °C. Peptides were extracted by 50 mM ABC buffer and acidified to pH < 2 by adding 10 μl of 10% TFA.
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