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Le220 ultrasonicator

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The LE220 ultrasonicator is a laboratory equipment designed for sample processing. It utilizes high-frequency sound waves to disrupt and homogenize materials, such as cells, tissues, and other samples. The LE220 is capable of generating precise and controlled acoustic energy to effectively process a wide range of sample types.

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Lab products found in correlation

Novaseq 6000, by Illumina (4 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions) Ampure xp bead, by Beckman Coulter (3 mentions) Qubit dsdna br assay, by Thermo Fisher Scientific (2 mentions) Sureselectxt2 human all exon v6 cosmic, by Agilent Technologies (2 mentions) Hiseq 4000, by Illumina (2 mentions) Unique dual indexed barcodes, by Integrated DNA Technologies (2 mentions) Sureselect target enrichment system, by Agilent Technologies (2 mentions) Kapa hifi kit, by Roche (2 mentions) Accuclear dsdna quantitation solution, by Biotium (2 mentions) Miseq nano kit, by Illumina (2 mentions) Hyperprep library preparation kits, by Roche (2 mentions) Quant it picogreen dsdna assay, by Thermo Fisher Scientific (2 mentions) Gentra puregene tissue kit, by Qiagen (2 mentions) Nucleospin tissue kit, by Takara Bio (1 mentions) Dna polymerase 1, by New England Biolabs (1 mentions) Minelute spin column, by Qiagen (1 mentions) Klenow fragment 3 5 exo, by New England Biolabs (1 mentions) Phusion hot start polymerase, by New England Biolabs (1 mentions) Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Ultrasonicator (113 citations) LE220 (72 citations) LE220 focused ultrasonicator (36 citations) LE220-plus Focused-ultrasonicator (16 citations) E220/LE220 Ultrasonicator (2 citations) LE220-plus ultrasonicator (2 citations)

17 protocols using le220 ultrasonicator

1

FFPE Whole Exome Sequencing Protocol

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Whole exome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumor tissue with matched germline DNA. Manual macrodissection was performed on FFPE slides, if necessary, using a scalpel and H&E stained slide as a guide. Tissue deparaffinization and DNA extraction were performed using standard methods. DNA was quantified using Qubit dsDNA BR Assay (Invitrogen) and sheared to an average of 250 bp with the Covaris LE220 ultrasonicator (Covaris Inc., Woburn, MA). For genomic library preparation, paired-end libraries were prepared using the NEBNext Ultra Kit (New England Biolabs, Ipswich, MA) and DNA library quality and fragment size were measured with an Agilent 2100 Bioanalyzer. Exome sequences were enriched from the genomic libraries according to the manufacturer’s protocol with the SureSelectXT2 Human All Exon V6+COSMIC (Agilent, Santa Clara, CA). Samples were pooled and sequenced on the Illumina HiSeq4000 with 150 bp paired end reads with an average target depth of 87x.
Huang A.C., Orlowski R.J., Xu X., Mick R., George S.M., Yan P.K., Manne S., Kraya A.A., Wubbenhorst B., Dorfman L., D’Andrea K., Wenz B.M., Liu S., Chilukuri L., Kozlov A., Carberry M., Giles L., Kier M.W., Quagliarello F., McGettigan S., Kreider K., Annamalai L., Zhao Q., Mogg R., Xu W., Blumenschein W.M., Yearley J.H., Linette G.P., Amaravadi R.K., Schuchter L.M., Herati R.S., Bengsch B., Nathanson K.L., Farwell M.D., Karakousis G.C., John Wherry E, & Mitchell T.C. (2019). A Single Dose of Neoadjuvant PD-1 Blockade Predicts Clinical Outcomes in Resectable Melanoma. Nature medicine, 25(3), 454-461.
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2

Amplification and Sequencing of ALS Samples

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After DNA extraction from control and ALS samples, the φ29-polymerase amplified DNA strands were sheared into fragments of 200 bp and ~ 350–400 bp in size using a Covaris LE220 ultrasonicator (Covaris, Brighton, UK) with operating parameters set as the following: 20 s ultrasound, acoustic duty factor 25%, peak incident power 500 W, cycles per burst 500, 24 cycles. Appropriate fragment size (200–500 bp) was captured with the aid of AMPure XP beads (Agencourt, Beverly, USA). Fragmented DNA was then incubated with bacteriophage T4 DNA polymerase (ENZYMATICS, Beverly, USA) for 30 min at 20 ℃ for trimming to obtain blunt ends devoid of 3′-overhangs or 5′-gaps, which were further 3′-adenlyated to create sticky ends. These DNA fragments were ligated at both ends to T-tailed adapters and amplified via PCR according to the following step-wise procedures: 3 min at 95 ℃ followed by 8 cycles of 20 s at 98 ℃; 15 s at 60 ℃; 30 s at 72 ℃; 10 min at 72 ℃ for further elongation. The obtained PCR products were purified via AMPure XP beads (Agencourt, Beverly). The DNA library was sequenced on BGISEQ-500 by paired-end sequencing of 150 bp intervals both from the forward and reverse strand, with an average sequencing data of 138 M reads per library, to provide high-quality alignment across all genomic regions.
Gerovska D., Noer J.B., Qin Y., Ain Q., Januzi D., Schwab M., Witte O.W., Araúzo-Bravo M.J, & Kretz A. (2023). A distinct circular DNA profile intersects with proteome changes in the genotoxic stress-related hSOD1G93A model of ALS. Cell & Bioscience, 13, 170.
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3

FFPE Whole Exome Sequencing Protocol

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Whole exome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) tumor tissue with matched germline DNA. Manual macrodissection was performed on FFPE slides, if necessary, using a scalpel and H&E stained slide as a guide. Tissue deparaffinization and DNA extraction were performed using standard methods. DNA was quantified using Qubit dsDNA BR Assay (Invitrogen) and sheared to an average of 250 bp with the Covaris LE220 ultrasonicator (Covaris Inc., Woburn, MA). For genomic library preparation, paired-end libraries were prepared using the NEBNext Ultra Kit (New England Biolabs, Ipswich, MA) and DNA library quality and fragment size were measured with an Agilent 2100 Bioanalyzer. Exome sequences were enriched from the genomic libraries according to the manufacturer’s protocol with the SureSelectXT2 Human All Exon V6+COSMIC (Agilent, Santa Clara, CA). Samples were pooled and sequenced on the Illumina HiSeq4000 with 150 bp paired end reads with an average target depth of 87x.
Huang A.C., Orlowski R.J., Xu X., Mick R., George S.M., Yan P.K., Manne S., Kraya A.A., Wubbenhorst B., Dorfman L., D’Andrea K., Wenz B.M., Liu S., Chilukuri L., Kozlov A., Carberry M., Giles L., Kier M.W., Quagliarello F., McGettigan S., Kreider K., Annamalai L., Zhao Q., Mogg R., Xu W., Blumenschein W.M., Yearley J.H., Linette G.P., Amaravadi R.K., Schuchter L.M., Herati R.S., Bengsch B., Nathanson K.L., Farwell M.D., Karakousis G.C., John Wherry E, & Mitchell T.C. (2019). A Single Dose of Neoadjuvant PD-1 Blockade Predicts Clinical Outcomes in Resectable Melanoma. Nature medicine, 25(3), 454-461.
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4

6mA Methylation Profiling in Oxytricha

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Genomic DNA was isolated from vegetative Oxytricha cells using the Nucleospin Tissue Kit (Takara Bio USA, Inc.). DNA was sheared into 150bp fragments using a Covaris LE220 ultra-sonicator (Covaris). Samples were gel-purified on a 2% agarose-TAE gel, blunted with DNA polymerase I (New England Biolabs), and purified using MinElute spin columns (QIAGEN). The fragmented DNA was dA-tailed using Klenow Fragment (3′ -> 5′ exo-) (New England Biolabs) and ligated to Illumina adaptors following manufacturer’s instructions. Subsequently, 2.2μg of adaptor-ligated DNA containing 6mA was immunoprecipitated using an anti-N6-methyladenosine antibody (Cedarlane Labs) conjugated to Dynabeads Protein A (Invitrogen). The anti-6mA antibody is commonly used for RNA applications, but has also been demonstrated to recognize 6mA in DNA (Fioravanti et al., 2013 (link); Xiao and Moore, 2011 ). The immunoprecipitated and input libraries were treated with proteinase K, extracted with phenol:chloroform, and ethanol precipitated. Finally, they were PCR-amplified using Phusion Hot Start polymerase (New England Biolabs) and used for Illumina sequencing.
Beh L.Y., Debelouchina G.T., Clay D.M., Thompson R.E., Lindblad K.A., Hutton E.R., Bracht J.R., Sebra R.P., Muir T.W, & Landweber L.F. (2019). Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization. Cell, 177(7), 1781-1796.e25.
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5

Whole-Genome Sequencing of PARK2 Carriers

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To dissect the novel GWAS signal associated with AAO identified in the Spanish population, we performed whole-genome sequencing (WGS) analyses in 5 of 37 homozygous carriers of the PARK2 signal. DNA concentration was determined by Qubit fluorescence and normalized to 20 ng/uL. One microgram of total genomic DNA was sheared to a target size of 450 base pairs (bp) using the Covaris LE220 ultrasonicator. Library preparation was achieved using the TruSeq DNA PCR-Free High Throughput Library Prep Kit and IDT for Illumina TruSeq DNA UD Indexes (96 Indexes, 96 Samples). Sequencing libraries were assessed for size distribution, absence of free adapters, and adapter dimers on a Fragment Analyzer. Library quantitation was performed by quantitative polymerase chain reaction using the KAPA Library Quantification Kit subsequent normalization to 4 nM. Libraries were clustered on v2.5 flowcell using the Illumina cBot 2 System before sequencing on the Illumina HiSeq X System using paired-end 150-bp reads. BCL files processed with alignment by ISAAC on HAS 2.2 and BAMs were used for QC assessment of mean coverage, percent duplicates, percent bases >20× coverage, and percent noise sites.
Bandres-Ciga S., Ahmed S., Sabir M.S., Blauwendraat C., Adarmes-Gómez A.D., Bernal-Bernal I., Bonilla-Toribio M., Buiza-Rueda D., Carrillo F., Carrión-Claro M., Gómez-Garre P., Jesús S., Labrador-Espinosa M.A., Macias D., Méndez-del-Barrio C., Periñán-Tocino T., Tejera-Parrado C., Vargas-González L., Diez-Fairen M., Alvarez I., Tartari J.P., Buongiorno M., Aguilar M., Gorostidi A., Bergareche J.A., Mondragon E., Vinagre-Aragon A., Croitoru I., Ruiz-Martínez J., Dols-Icardo O., Kulisevsky J., Marín-Lahoz J., Pagonabarraga J., Pascual-Sedano B., Ezquerra M., Cámara A., Compta Y., Fernández M., Fernández-Santiago R., Muñoz E., Tolosa E., Valldeoriola F., Gonzalez-Aramburu I., Sanchez Rodriguez A., Sierra M., Menéndez-González M., Blazquez M., Garcia C., Suarez-San Martin E., García-Ruiz P., Martínez-Castrillo J.C., Vela-Desojo L., Ruz C., Barrero F.J., Escamilla-Sevilla F., Mínguez-Castellanos A., Cerdan D., Tabernero C., Gomez Heredia M.J., Perez Errazquin F., Romero-Acebal M., Feliz C., Lopez-Sendon J.L., Mata M., Martínez Torres I., Kim J.J., Dalgard C.L., Brooks J., Saez-Atienzar S., Gibbs J.R., Jorda R., Botia J.A., Bonet-Ponce L., Morrison K.E., Clarke C., Tan M., Morris H., Edsall C., Hernandez D., Simon-Sanchez J., Nalls M.A., Scholz S.W., Jimenez-Escrig A., Duarte J., Vives F., Duran R., Hoenicka J., Alvarez V., Infante J., Marti M.J., Clarimón J., López de Munain A., Pastor P., Mir P, & Singleton A. (2019). The Genetic Architecture of Parkinson Disease in Spain: Characterizing Population-Specific Risk, Differential Haplotype Structures, and Providing Etiologic Insight. Movement disorders : official journal of the Movement Disorder Society, 34(12), 1851-1863.
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6

Whole-exome sequencing for genetic diagnosis

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We collected approximately 2~3 ml of peripheral blood from the boy (EDTA anticoagulation), and then peripheral blood DNA was extracted by using a Blood DNA Midi Kit (Omega D3494-04, Biotek, USA). To make a precise diagnosis, we performed WES on the proband. Using 3 µg of genomic DNA from each subject for detection, we sheared the DNA into 100~500 base pairs (bp) with a Covaris LE220 ultrasonicator (Massachusetts, USA), and then fragments measuring 150 to 200 bp were picked out by magnetic beads. An adaptor-ligated library that was quality controlled by an Agilent 2100 Bioanalyzer was set up for each individual subject. The cyclized library was continuously sequenced for 50 cycles by a BGISEQ-500 high-throughput sequencer (BGI, Shenzhen, China), and the raw sequencing data were read. Sequencing reads were aligned to a reference human genome (HG19/HG20) using Burrows-Wheeler Aligner (BWA) software, and single nucleotide variants and insertions and deletions were detected by Genome Analysis Toolkit 4.0 (GATK) software, followed by alignment of the database (NCBI dbSNP, HapMap, the 1000 Genomes dataset and a database of 100 healthy Chinese adults) screened for suspicious variants.
Li R., Tian B., Liang H., Chen M., Yang H., Wang L., Pan H, & Zhu H. (2021). A Chinese Case of Cornelia de Lange Syndrome Caused by a Pathogenic Variant in SMC3 and a Literature Review. Frontiers in Endocrinology, 12, 604500.
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7

Optimizing Spike-in Control DNA for cfMeDIP-seq

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We determined the optimal amount of spike-in control DNA needed per experiment by adding varying amounts of spike-in controls to sheared mycoplasma-free HCT116 genomic DNA (American Type Culture Collection, Manassas, VA, USA, RRID: CVCL_0291). Optimizing the amount of spike-in control DNA added to an experiment avoids using a large portion of the sequencing reads on spike-in fragments, saving most reads for the biological sample. We sheared the HCT116 genomic DNA using an LE220 ultrasonicator (Covaris, Woburn, MA, USA). Using AMPure XP beads (Beckman Coulter, Brea, CA, USA), we size selected to ∼150 bp in length to mimic cfDNA input. We created 3 replicate samples of sheared HCT116 cfDNA mimic with masses of synthetic spike-in control DNA of 0.1 ng, 0.05 ng, and 0.01 ng. We performed the cfMeDIP-seq experiment as previously described. 2 Samples underwent sequencing (Princess Margaret Genomics Centre, Toronto, ON, Canada) on a NovaSeq 6000 (Illumina, San Diego, CA, USA), paired-end 2×100 bp, 60 million paired-end reads per sample (Figure 1).
Wilson S.L., Shen S.Y., McKinnon L.M., Burgener J., Triche T., Bratman S.V., Carvalho D.D.D., & Hoffman M.M. (2021). Sensitive and reproducible cell-free methylome quantification with synthetic spike-in controls.
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8

ChIP-Seq Protocol for Transcription Factor and Histone Modifications

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Visualization and analysis of existing ATAC-Seq and ChIP-Seq data are described in Supplemental Methods. Chromatin immunoprecipitation in HoxB-FL cells was performed as previously described (Ghosh et al., 2014 (link)). Briefly, 3×106 cells were fixed for 5 min using the truChIP™ High Cell Chromatin Shearing Kit with SDS Shearing Buffer (Covaris) according to manufacturer’s protocol. The fixed nuclei were sheared using the Covaris LE220 ultrasonicator for 6 minutes. Sheared chromatin was incubated overnight at 4°C with magnetic Protein A beads pre-coated with Abs to TCF4, H3K4me1 (Abcam) or H3K27ac (Abcam). The DNA was eluted from beads by incubating at 65°C for 15 min, incubated overnight at 65°C to reverse crosslinks, purified and used for qPCR using primers listed in Supplemental Methods. A fixed amount of total chromatin input was used as a control. Percent input was calculated using 100*2^(adjusted input - CT(IP), where adjusted input equals CT(input)-log2(dilution factor). Fold enrichment was calculated using % input (region of interest) divided by % input (control region).
Grajkowska L.T., Ceribelli M., Lau C.M., Warren M.E., Tiniakou I., Higa S.N., Bunin A., Haecker H., Mirny L.A., Staudt L.M, & Reizis B. (2016). Isoform-specific expression and feedback regulation of E protein TCF4 control dendritic cell lineage specification. Immunity, 46(1), 65-77.
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9

Crosslinking and Chromatin Sonication

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K562 cells were counted before crosslinking. 250 × 103, 125 × 103, 62 × 103, and 31 × 103 cells were crosslinked in 1 % formaldehyde for 10 min at room temperature. After quenching with glycine and washing in cold PBS, cells were lysed in 1 ml of lysis buffer (50 mM HEPES KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10 % glycerol, 0.5 % Nonidet P-40, and 0.25 % Triton X-100 supplemented with protease inhibitors). Nuclei were washed in 1 ml of washing buffer (10 mM Tris pH 8, 200 mM NaCl, 1 mM EDTA pH 8, 0.5 mM EGTA, supplemented with protease inhibitors) followed by a rinse in TE buffer supplemented with protease inhibitor. Nuclei were counted to verify the amount per each sample and resuspended in 135 μl of TE buffer supplemented with 0.1 % Triton X-100 and protease inhibitors. Nuclei were sonicated using Covaris LE220 ultrasonicator for 20, 25 and 30 min. Debris were removed and an aliquot of chromatin for each samples underwent reverse crosslinking at 65 °C O/N. Afterwards, DNA was purified using 24:1 chloroform/isoamyl alcohol (PCI). DNA was then quantified using Qubit HS dsDNA assay (Life Technologies) and fragmentation size distribution was tested using Agilent High Sensitivity DNA Assay.
Valensisi C., Liao J.L., Andrus C., Battle S.L, & Hawkins R.D. (2015). cChIP-seq: a robust small-scale method for investigation of histone modifications. BMC Genomics, 16, 1083.
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10

Comprehensive Genomic Sequencing Protocol

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Extracted genomic DNA was sheared to 100-400 bp (mean distribution 150 bp) using an LE220 ultrasonicator (Covaris Inc). Libraries were prepared (NEBNext Ultra II DNA Library prep Kit, New England Biolabs, Massachusetts, USA) using initial adaptor ligation and barcoding with unique dual indexed barcodes (Integrated DNA Technologies, Iowa, USA). Dual indexed samples were amplified (6 cycles of PCR, KAPA HiFi kit, Roche, Basel, Switzerland), quantified (Accuclear dsDNA Quantitation Solution, Biotium, California, USA), then pooled in preassigned groups of 48 or 32 to generate equimolar pools based on Total DNA concentration. 500 ng pooled DNA was hybridised using 120-mer RNA baits (SureSelect Target enrichment system, Agilent technologies; Bait design ELID ID 0616571)4 (link),37 (link). Enriched libraries were sequenced on Illumina HiSeq 4000 to generate 150 bp paired end reads at the Wellcome Sanger Institute (Cambridgeshire, UK) as previously described38 . For one rabbit passaged sample from Melbourne, Australia (TPA_AUSMELT-1)30 , genomic DNA extracted from historically archived tissue lysate was sequenced on Illumina NextSeq 500 (150 bp paired end reads, Nextera DNA Flex libraries) without any prior enrichment to an estimated 1Gb/sample at the Doherty Institute (Melbourne, Australia).
Beale M.A., Marks M., Cole M.J., Lee M.K., Pitt R., Ruis C., Balla E., Crucitti T., Ewens M., Fernández-Naval C., Grankvist A., Guiver M., Kenyon C.R., Khairullin R., Kularatne R., Arando M., Molini B.J., Obukhov A., Page E.E., Petrovay F., Rietmeijer C., Rowley D., Shokoples S., Smit E., Sweeney E.L., Taiaroa G., Vera J.H., Wennerås C., Whiley D.M., Williamson D.A., Hughes G., Naidu P., Unemo M., Krajden M., Lukehart S.A., Morshed M.G., Fifer H, & Thomson N.R. (2021). Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis. Nature Microbiology, 6(12), 1549-1560.
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