Ultrasonic DNA Shearing
- During DNA and RNA shearing, DNA molecules are broken into smaller pieces. DNA / RNA fragmentation is one of the important sample prep steps required create libraries for next generation sequencing (NGS).
- Ultrasonic DNA shearing uses the forces of acoustic cavitation to break the DNA or RNA into pieces of 100 – 5kb bp.
- Ultrasonic shearing allows for precise DNA fragmentation and adaption to the desired DNA length.
Ultrasonic DNA Shearing
Hielscher Ultrasonics offers various ultrasound-based solutions for DNA, RNA and chromatin shearing. Choose between a probe-type ultrasonicators (e.g. UP100H) for direct sonication using a microtip, or use the VialTweeeter or the ultrasonic cuphorn for indirect DNA preparation of various samples simultaneously. Hielscher offers the ideal device considering your needs: wether you have 1 or up to 10 samples, volumes from microliter to liter volumes – Hielscher ultrasonic processors are available to meet your requirements to prepare DNA, RNA and chromatin fragments at the right length. Reproducibility, easy operation and precise control allow for a reliable library for next-generation sequencing.
In contrast to enzymatic DNA fragmentation, ultrasonic shearing applies pure mechanical shear forces without adding any chemicals. By the precise setting of process parameters, ultrasonic shearing produces high molecular weight DNA fragments (plasmid and genomic DNA).
Purified nucleic acids can be amplified prior to or after a fragmentation step.
Sonication parameters (power, pulse cycle / bursts, time and temperature) can be controlled safely via software settings.
Protocols for Ultrasonic DNA Shearing
For Chromatin Immunoprecipitation Assay
Briefly, cells were plated in 60mm-diameter dishes (400,000 per dish) and transfected with RhoA siRNA (as described); after 72 h, they were incubated with formaldehyde (final concentration, 1%) for 10 min at 37°C to cross-link proteins to DNA. The cross-linking reaction was quenched by the addition of one-tenth volume of 1.25 mol/L glycine, giving 125 mmol/L final concentration. Cells were washed twice with ice-cold PBS, resuspended in radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 5 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 8.0)] containing 1 mmol/L phenylmethylsulfonyl fluoride, 1 Ag/mL aprotinin, and 1 Ag/mL pepstatin A, and kept on ice for 30 min. Then, cell lysates were sonicated on ice with a Hielscher UP200S ultrasound sonicator (3 x 40 s, amplitude 40%, cycle 1; Hielscher Ultrasonics GmbH) until cross-linked chromatins were sheared to yield DNA fragments between 200 and 1,000 bp. One tenth of whole lysate was used to quantitate the amount of DNA present in different samples and considered as “total input DNA”. Supernatants were incubated with salmon sperm DNA/protein agarose-50% slurry to reduce nonspecific background. Immunoprecipitation was then done overnight at 4°C with 5 Ag of anti-NF-nB p65 (Upstate) or without antibody (negative control). These supernatants were supplemented with 5 mol/L NaCl and heated overnight at 65°C to revert protein-DNA cross-links. The immunocomplexes were further treated with DNase- and RNase-free proteinase K, and DNA was purified by phenol/chloroform extraction and ethanol precipitation. PCR was done with specific primers corresponding to a sequence within the promoter region of the human iNOS gene (p1 primer: 5¶-GAGGGCTTTCCCA- GAACCAAG-3¶; p2 primer: GCTGGGCTACTGACCCAG- CAGTTCCAG-3¶). (Doublier et al., 2008)
For expression studies, the recombinant strain L. tarentolae p10::F9Begfp1.4dBsat#12 (Jena Bioscience, Germany) with the gene for EGFP (Enhanced Green Fluorescent Protein), chromosomal ssu integrated, was cultivated in the various media as described previously and additionally supplemented with 100 mg l-1 Nourseothricin (Jena Bioscience, Germany). During cultivation, 1 ml samples were taken, centrifuged (2000 × g, 20°C, 10 min) and washed with 0.9% NaCl solution. Pellet was resuspended in buffer (20 mM HEPES, 5 mM EDTA, 2 mM DTT) and disintegrated by sonification with the ultrasonic processor UP400S (application of energy ∼ 400 Ws). Cell debris were removed by centrifugation (6000 × g, 4°C, 5 min) and analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions according to the method of Laemmli (1970) with 12.5% polyacralamide gels. EGFP-expression was examined in agitated culture. (Fritsche et al. 2007)
The chromatin immunoprecipitation assay was performed using the ChIP-ITTM Express (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions with some modifications. Briefly, differentiated human podocytes were cross-linked with 1% formaldehyde for 10 min at room temperature. Cells were washed with ice-cold PBS and the fixation reaction was stopped by adding 0.125 M glycine for 5 min at room temperature. Cells were washed again with ice-cold PBS and scraped from the dish. Cells were pelleted by centrifugation and resuspended in the lysis buffer. After centrifugation, pelleted nuclei were resuspended in the shearing buffer, incubated on ice for 30 min and the chromatin was sheared by sonication, e.g. UP100H (Hielscher Ultrasonics GmbH, Teltow, Germany) at 25% power 5 pulses of 20 sec each on ice into fragments of approximately 200–600 bp. The sheared chromatin was then centrifuged and the supernatant was collected. For immunoprecipitations, 60 μl of chromatin was incubated with 1 μg of Sp1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), NF-κB p65 (Abcam, Cambridge, UK) or NF-κB p50 (Abcam) antibodies or with rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA), as a negative control, overnight at 4°C with gentle rotation. Immunocomplexes bound to magnetic beads were collected using a magnetic stand, washed extensively, and the protein/DNA crosslinks were reversed and DNA eluted for real-time PCR analysis. (Ristola et al. 2009)
EHEC DNA preparation for chip array analysis
Arrangement of cell lysates and extracted DNAs
Bacterial pellets suspended in PBS to the desired final concentration were treated with ultrasound disruptor UP100H (Hielscher GmbH, Germany) equipped with a microtip MS1 (1mm in diameter). The operating frequency was 30 kHz and effective output power was 100 W. During the operation, samples were cooled in an ice-water bath, mixed and centrifuged. The samples were utilized for flow cytometry studies, while for later handling, samples were subjected to a heat treatment (95°C, 5 min). The crude cell lysates were processed with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1). An equal volume of this mix was added to the lysate sample, the solution was vortexed vigorously for 15 s and centrifuged at 15,000 x g for 2 min at room temperature (RT) around 22°C. The top aqueous phase containing the genomic DNA was carefully separated and collected in a new sterile Eppendorf tube.
Subsequently, samples were sonicated to fragment the DNA. The sonication step was realized in the same conditions as described above. To evaluate the fragmentation effects on the genomic DNA, samples were analyzed by using agarose gel electrophoresis.
(…) The samples sonicated previously for 2.5 min were subjected to an extraction step after heat treatment and centrifugation. DNA released was extracted two times with a phenol:chloroform:isoamyl alcohol mix, and afterwards subjected to second sonication for 0 – 15 min. Agarose gel electrophoresis was used to determine the size distribution of DNA subjected to post-extraction ultrasonic fragmentation (Fig. at the top right side). Highly fragmented DNA was evident from the presence of a DNA smear rather than high-molecular weight bands that were eliminated from samples sonicated for 2.5 min or longer. Longer sonication gradually reduced fragment lengths to approximately 150 – 600 bp, and sonication for 15 min further degraded these fragments, as can be seen mostly by the upper part of the smear. Thus, the average DNA fragment size gradually declined with ultrasonication time and the 5 min treatment allowed to obtain the sizes of DNA fragments most suitable for chip array assays. At last, the DNA analyte preparation procedure comprising first 2 min of ultrasonic treatment, DNA extraction (2×), and subsequent 5 min sonication, was established. (Basselet et al. 2008)
Chromatin Immunoprecipitation (ChIP)
HEK293 cells were cultured as described above and fixed with 2 mM disuccinimidyl-glutarate for 45 min at room temperature. Subsequently, the cells were washed twice with PBS. Chromatin was cross-linked for 10 min at room temperature using 1% (v/v) formaldehyde and washed twice with ice-cold PBS. The cross-linking reaction was stopped by incubation with glycine at a final concentration of 0.125 M for 5 min at room temperature. After incubation with trypsin, the cells were scraped from the cell culture dish and washed twice with PBS. The cell pellet was resuspended in lysis buffer (5 mM Pipes, pH 8.0, 85 mM KCl, and 0.5% (v/v) Nonidet P-40), incubated on ice for 10 min, and homogenized with a Dounce homogenizer. Subsequently, nuclei were pelleted by centrifugation (3500 x g, 5 min, 4 °C) and resuspended in nuclei buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, and 1% (w/v) SDS). Nuclei were disrupted by sonication with three 20-s pulses in a UP50H sonicator (Hielscher Ultraschall Technologie) at a setting of cycle 0.5 and amplitude 30%, yielding genomic DNA fragments with a bulk size of 200 – 1000 bp. For ChIP, 50g of DNA was diluted 4-fold in immunoprecipitation buffer (16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% (v/v) Triton X-100, and 0.01% (w/v) SDS). (Weiske et al. 2006)
Histone modification analysis by chromatin immunoprecipitation (ChIP)
Briefly, 6 x 106 cells were washed twice with PBS and cross-linked on the culture plate for 15 min at room temperature in the presence of 0.5% formaldehyde. Cross-linking reaction was stopped by adding 0.125 M glycine. All subsequent steps were carried out at 48°C. All buffers were pre-chilled and contained protease inhibitors (Complete Mini, Roche). Cells were washed twice with PBS and then scraped. Collected pellets were dissolved in 1 ml lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris pH 8) and were sonicated in a cold ethanol bath for 10 cycles at 100% amplitude using a UP50H sonicator (Hielscher, Teltow, Germany). Chromatin fragmentation was visualized in 1% agarose gel. Obtained fragments were in the 200–500pb range. Soluble chromatin was obtained by centrifuging the sonicated samples at 14,000g for 10 min at 48°C. The soluble fraction was diluted 1/10 in dilution buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8, 150 mM NaCl) then aliquoted and stored at 80°C until use. (Rodriguez et al. 2008)
|Device||Power [W]||Type||Volume [mL]|
|UP50H||50||handheld or standmounted||0.01||–||250|
|UP100H||100||handheld or standmounted||0.01||–||500|
|UP200Ht||200||handheld or standmounted||0.1||–||1000|
|GDmini2||200||contamination-free flow cell|
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Ultrasonic / acoustic cavitation creates highly intense forces which opens the cell walls known as lysis (Click to enlarge!)
- Basselet P., Wegrzyn G., Enfors S.-O., Gabig-Ciminska M. (2008): Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC). Microbial Cell Factories 7:29. 2008.
- Doublier S., Riganti Ch., Voena C., Costamagna C., Aldieri E., Pescarmona G., Ghigo D., Bosia A. (008): RhoA Silencing Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells. Molecular Cancer Research 6(10), 2008.
- Fredlund E., Gidlund A., Olsen M., Börjesson T., Spliid N.H.H., Simonsson M. (2008): Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels. Journal of Microbiological Methods 2008.
- Fritsche C., Sitz M., Weiland N., Breitling R., Pohl H.-D. (2007): Characterization of the growth behavior of Leishmania tarentolae – a new expression system for recombinant proteins. Journal of Basic Microbiology 47, 2007. 384–393.
- Ristola M., Arpiainen S., Saleem M. A., Mathieson P. W., Welsh G. I., Lehtonen S., Holthöfer H. (2009): Regulation of Neph3 gene in podocytes – key roles of transcription factors NF-κB and Sp1. BMC Molecular Biology 10:83, 2009.
- Rodriguez J., Vives L., Jorda M., Morales C., Munoz M., Vendrell E., Peinado M. A. (2008): Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells. Nucleic Acids Research Vol. 36, No. 3, 2008. 770-784.
- Weiske J. Huber O. (2006): The Histidine Triad Protein Hint1 Triggers Apoptosis Independent of Its Enzymatic Activity. The Journal of Biological chemistry. Vol. 281, No. 37, 2006. 27356–27366.