Genomic DNA quality was determined by estimating the A260/A280 and A260/A230 ratios by nanodrop and concentration by Qubit dsDNA BR Assay. DNA integrity and size was confirmed by running an Agilent Bioanalyzer gel. Genomic DNA (1 µg) was sheared to a fragment length of 150–200 bp using a focused acoustic energy Covaris E220 system (Covaris, part#500003). Fragmented sample size distribution was determined by the Caliper LabChip GX system (PerkinElmer, Part#122000). Unmethylated C residues of enriched DNA were modified by bisulfite conversion using EZ DNA Methylation-Gold Kit (Zymo Research, part#D5005). Indexed paired-end whole genome sequencing libraries are prepared using SureSelect XT Methyl-Seq kit (Agilent, part#G9651B).
E220 system
Manufactured by Covaris
Sourced in United States
The E220 system is a compact and powerful benchtop instrument designed for DNA, RNA, and protein extraction and preparation. It utilizes Covaris' proprietary Adaptive Focused Acoustics (AFA) technology to perform non-contact, isothermal sample processing with exceptional reproducibility. The E220 system is capable of performing a wide range of applications, including sample lysis, homogenization, and fragmentation.
Automatically generated - may contain errors
Lab products found in correlation
Caliper labchip gx system, by PerkinElmer (2 mentions)
Truseq dna pcr free kit, by Illumina (2 mentions)
Truseq dna cd indexes, by Illumina (2 mentions)
Ampure xp bead, by Illumina (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer gel, by Agilent Technologies (1 mentions)
Sureselect xt methyl seq kit, by Agilent Technologies (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Amp pure beads, by Beckman Coulter (1 mentions)
Rnase a, by Roche (1 mentions)
Qrt pcr, by Roche (1 mentions)
Idt xgen exome research panel v1, by Integrated DNA Technologies (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
Nebnext enzymatic methyl seq kit, by New England Biolabs (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000 system, by Novogene (1 mentions)
Dneasy plant pro kit, by Qiagen (1 mentions)
Paired end libraries, by Illumina (1 mentions)
13 protocols using e220 system
1
Whole Genome Bisulfite Sequencing
2
Chromatin Immunoprecipitation Protocol
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For chromatin immunoprecipitation (ChIP) experiments, prepared cells were collected following 48 h of lentiviral infection and 5-d selection (unless otherwise indicate) with puromycin or blasticidin. Capture of chromatin-bound proteins was performed using standard protocols (Millipore). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at 37 °C, the reaction was quenched by addition of 125 mM glycine for 5 min and then five (for synovial sarcoma cell lines) or ten (for fibroblast cell lines) million fixed cells were used per experiment. Chromatin was fragmented by sonication with a Covaris E220 system and the solubilized chromatin was incubated with a primary antibody overnight at 4 °C to form antibody–chromatin complexes. These complexes were incubated with protein G Dynabeads (Thermo Scientific) for 3 h at 4 °C then the beads were washed three times and eluted. The samples then underwent crosslink reversal, treatment with RNase A (Roche) and treatment with proteinase K (Thermo Scientific) followed by DNA capture with AMP Pure beads (Beckman Coulter).
3
Exome Sequencing of TN4 Family
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Genomic DNA was extracted from whole blood of 11 cases and two unaffected siblings (a brother and a sister) of TN4 (TN1-10 and TN12) with informed consent using NucleoSpin Tissue kit (#740952, Machery-Nagel, Duren, Germany) according to the manufacturer’s instructions. Exome library preparation and sequencing were performed by the Yale Center for Genome Analysis. Briefly, 1 µg of genomic DNA was sheared into fragments with the Covaris E220 system (Covaris, Woburn, MA, United States), followed by end-repair, A-tailing, ligation of multiplexing adaptors, and pre-capture ligation-mediated PCR amplification. The quantification and insert size distribution of product were determined using Caliper LabChip GX system (PerkinElmer Inc., Santa Clara, CA, USA). Exome library was captured using IDT xGen Exome Research Panel V1.0 (Integrated DNA Technologies, Coralville, IA, USA), and quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Wilmington MA, USA). Libraries were then loaded onto S4 flowcell and sequenced on the Illumina NovaSeq6000 platform using 101 bp paired-end sequencing reads according to Illumina protocols (Illumina Inc., San Diego, CA, USA).
4
Enzymatic Methylation-Sequencing Protocol
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Approximately 5 ng of genomic DNA was extracted from ∼1.5 × 105 nuclei of each type of sample, using DNeasy Plant Pro Kit (QIAGEN), and treated with RNase A (Thermo Scientific) to remove any contaminating RNA. Subsequently, ∼5 ng DNA (including 0.02 ng of unmethylated lambda DNA and 0.001 ng of >96% methylated pUC19 DNA spiked in) was sheared to 100–500-bp fragments using a focused ultrasonicator (Covaris E220 system), in microtubes at a setting of 175 peak incident power, 10 dc, 200 cpb for 40 s. Enzymatic methyl-sequencing (EM-seq) libraries were prepared from sheared DNA using NEBNext Enzymatic Methyl-seq Kit following the manufacturer instructions (New England BioLabs). Due to the amount of starting DNA material being lower than 10 ng, the minimum recommended amount of starting material by the manufacturer, we added two PCR cycles at the final step of PCR amplification of the sequencing libraries. Libraries were sequenced on NovaSeq 6000 system (Novogene) for collecting 2× 150-bp paired-end reads that passed the Illumina quality control filter (Supplementary Table 8 ).
5
Histone and Nonhistone Protein ChIP
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ChIP was performed as described previously (Lee et al. 2006 (link)). Samples were sonicated using a Covaris E220 system. Sonication conditions were 20% duty factor for histones or 10% duty factor for nonhistone proteins, peak intensity pulse 140, and 200 cycles per burst, for 4 min.
6
Whole Genome Sequencing of Melanoma Cell Lines
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The Illumina paired-end libraries (Illumina, Inc.) techniques were used to detect and design the entire genome library and sequencing, as previously explained [30 (link)]. The amplified DNA (900 ng to 1 μg) from the advanced melanoma cell lines C8161, MUM-2B, and SK-MEL-28 was sheared into fragments of about 300 bp using a Covaris E220 system(Covaris Inc.). An aliquot of adaptor-ligated DNA was generated to use in the enrichment qPCR to establish the adaptor for Illumina PE sequencing. The pooled DNA (600 ng) from four barcoded libraries was utilized for hybridization and post-hybridization amplification according to the manufacturer’s instructions (SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library; Illumina, Inc.). The results of the PCR amplification of cell lines were quality confirmed, and Illumina HiSeq 2000 (Illumina, Inc.) was used for WGS, with an average coverage depth of between 10x and 30x.
7
FFPE DNA Illumina Library Preparation
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FFPE DNA samples were constructed into Illumina paired-end pre-capture libraries according to the 500 ng or 250 ng (depending on sample quantity availability) of DNA in 50ul volume. gDNA was sheared into fragments of the average size of 250–300 base pairs in a Covaris plate with an E220 system (Covaris, Inc. Woburn, MA) followed by end-repair (NEBNext End Repair Module—E6050L) and A-tailing (NEBNext dA-Tailing Module—E6053L), ligated with Illumina Dual index PE adapter using (Life Tech. ExpressLink Ligase- A13726101), Pre-capture Ligation Mediated-PCR (LM-PCR) was performed for 6–8 cycles using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc., Cat # KK2612). Universal P1.1 (5′-AATGATACGGCGACCACCGAGA) and P3 (5′-CAAGCAGAAGACGGCATACGAGA) primers were used to amplify the ligated products. AMPure XP beads (A63882, Beckman Coulter) were used for library purification. Quantification and size distribution of the pre-capture LM-PCR product were determined using the Fragment Analyzer capillary electrophoresis system (Agilent Technologies, Inc).
8
Chromatin Immunoprecipitation and Quantification
9
Automated Illumina Library Preparation
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Illumina libraries were prepared using an automated version of the TruSeq DNA PCR-Free kit. Briefly, DNA was quantified using Qubit HS DNA, and 1 μg of DNA was used as input. The samples were then fragmented using Covaris E220 system, aiming for a fragment size of 350 bp. Fragmented DNA was end-repaired, followed by size selection using Dynabeads MyOne Carboxylic Acid beads. Illumina TruSeq DNA CD Indexes with sample-specific barcode sequences were ligated and the final product was cleaned up using AMPure XP beads. Finished libraries were normalized based on their concentration and sequenced on NovaSeq6000 (NovaSeq Control Software 1.6.0/RTA v3.4.4) with a 2 × 151 setup using ‘NovaSeqXp’ workflow in ‘S4’ mode flowcell. Bcl to FastQ conversion was performed using bcl2fastq_v2.20.0.422 from the CASAVA software suite (v1.6).
10
Genomic DNA Isolation and Sequencing of Saro-13
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The virulent strain of S. oryzae (Saro-13) was grown on potato dextrose broth (PDB) medium for three days. The mycelium was grinded using liquid nitrogen and genomic DNA was isolated using nucleo-pore gDNA fungal and bacterial mini kit (Genaxy, Catalogue# NP-7006D). The DNA quality and quantity was assessed by Nanodrop and Qubit (Applied Biosystems), respectively. The genomic DNA was sheared to generate fragments of approximately 400–600 bp in Covaris microtube with the E220 system (Covaris, Inc., Woburn, MA, USA). The fragment size distribution was checked using Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) with high sensitivity DNA Kit (Agilent Technologies). The fragmented DNA was cleaned up using HighPrep beads (MagBio Genomics Inc, Gaithersburg, Maryland). The Illumina paired-end library was prepared as per manufacturers instruction using NEXTflex DNA sequencing Kit (Catalogue # 5140–02, Bioo Scientific). The paired end library was sequenced using Illumina NextSeq 500 in Genotypic Technologies, Bengaluru and the length of read sequence was 151 nts from both the ends of the fragment.
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