Chirp was performed as described by Chu et al 18 . Briefly, mouse livers were cross-linked using glutaraldehyde. After glycine quenching, the nuclear lysate was sonicated for 25–30 cycles 30 seconds on 30 seconds off at 4°C with BioRuptor twin sonicator (Diagenode). LeXis and LacZ pulldown probes with BiotinTEG at 3’ were designed by Biosearch Tecnologies (see supplementary information ) and allowed to hybridize overnight with sonicated chromatin at 37 °C (100 pmol probe per 1 mL chromatin). Following hybridization, C1 Dynabeads (Life Technologies) were added and incubated for 30 minutes. For protein elution for mass spectrometry analysis, washed beads were resuspended in 3× original volume of DNase buffer (100 mM NaCl and 0.1% NP-40), and protein was eluted with a cocktail of 50 mM triethyl ammonium bicarbonate, 12 mM sodium lauryl sarcosine, and 0.5% sodium deoxycholate supplemented with 100 ug/ml RNase A (Sigma-Aldrich) and 0.1 Units/microliter RNase H (Epicenter), and 100 U/ml DNase I (Invitrogen). For RNA isolation, beads were resuspended in protensase K buffer (100 mM NaCl, 10 mM TrisCl pH 7.0, 1 mM EDTA, 0.5% SDS, 5% by volume Proteainse K Ambion AM2546 20 mg/ml) and incubated at 50 °C followed by Trizol isolation and DNase treatment.
Bioruptor twin sonicator
Manufactured by Diagenode
Sourced in Belgium
The BioRuptor twin sonicator is a laboratory instrument designed for the efficient fragmentation of biological samples. It uses high-frequency ultrasonic waves to disrupt and shear DNA, RNA, and protein samples. The BioRuptor twin provides simultaneous processing of up to 12 samples in a single run, ensuring consistent and reproducible results.
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Lab products found in correlation
4 protocols using bioruptor twin sonicator
1
Chromatin Interaction Capture by Hybridization
2
Chromatin Interaction Capture by Hybridization
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Chirp was performed as described by Chu et al 18 . Briefly, mouse livers were cross-linked using glutaraldehyde. After glycine quenching, the nuclear lysate was sonicated for 25–30 cycles 30 seconds on 30 seconds off at 4°C with BioRuptor twin sonicator (Diagenode). LeXis and LacZ pulldown probes with BiotinTEG at 3’ were designed by Biosearch Tecnologies (see supplementary information ) and allowed to hybridize overnight with sonicated chromatin at 37 °C (100 pmol probe per 1 mL chromatin). Following hybridization, C1 Dynabeads (Life Technologies) were added and incubated for 30 minutes. For protein elution for mass spectrometry analysis, washed beads were resuspended in 3× original volume of DNase buffer (100 mM NaCl and 0.1% NP-40), and protein was eluted with a cocktail of 50 mM triethyl ammonium bicarbonate, 12 mM sodium lauryl sarcosine, and 0.5% sodium deoxycholate supplemented with 100 ug/ml RNase A (Sigma-Aldrich) and 0.1 Units/microliter RNase H (Epicenter), and 100 U/ml DNase I (Invitrogen). For RNA isolation, beads were resuspended in protensase K buffer (100 mM NaCl, 10 mM TrisCl pH 7.0, 1 mM EDTA, 0.5% SDS, 5% by volume Proteainse K Ambion AM2546 20 mg/ml) and incubated at 50 °C followed by Trizol isolation and DNase treatment.
3
MeDIP-Seq Library Preparation from CVS and Plasma
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DNA from CVS and non-pregnant female plasma were used to generate libraries using standard preparation methods. Genomic DNA obtained from CVS samples ranging from 12–30 ng was sheared to an average size of 230bp using the Bioruptor Twin Sonicator (UCD400, Diagenode, Liege, Belgium) and run on the TapeStation 2200 (Agilent Technologies, Santa Clara, CA USA) for fragment size verification. Blunt-ending and sequencing-adaptor ligation were performed prior to MeDIP using NEB Blunting and Ligase enzymes (NEB, Ipswich, UK) as previously described [16 ,24 ,25 (link)]. For synthetic pregnancy samples and pregnancy cases, library preparation was performed using the iDEAL Library Preparation kit (Diagenode) following the manufacturer’s protocol. MeDIP was performed as described previously for the immunoprecipitation of hypermethylated DNA [16 ,26 (link)]. Sequencing libraries of CVS and non-pregnant plasma samples were amplified for 30 cycles following MeDIP.
4
Fibrillarin Protein Extraction and Quantification
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Approximately 1 × 106 cells were scraped in 1 ml of cold phosphate buffered saline (PBS) and centrifuged for 5΄ at 1000g. Cell pellets were resuspended in 200 ml of cold F-Buffer [10 mM Tris–HCl (pH 7.0), 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride and 5 mM ZnCl2]. Cells were subjected to three cycles of sonication (30 s ON, 30 s OFF, high power) using a Bioruptor Twin sonicator (Diagenode), and then stored on ice for 10’. Cell extract was then centrifuged for 10 min at 14 000g, and the pellet was discarded. Cell lysates were quantified using BCA Protein Assay Kit ( Pierce), and 15 μg of total proteins were loaded on each lane of a 4–20% polyacrylamide gel.
Rabbit monoclonal antibody against Fibrillarin was obtained from Cell Signaling (cat. #2639), and used at a final dilution of 1:1000. Beta-actin was used as the loading control.
Rabbit monoclonal antibody against Fibrillarin was obtained from Cell Signaling (cat. #2639), and used at a final dilution of 1:1000. Beta-actin was used as the loading control.
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