DNA was extracted for sequencing using the Qiagen Blood & Tissue Kit (Cat. No. 69506). Prior to library preparation 2500 ng of gDNA were sheared with Covaris ME220 (Covaris, Inc.) to a mean fragment size of 550bp. Library preparation was performed using the VAHTS Universal DNA Library Prep Kit for Illumina (Vazyme Biotech Co.; Ltd) according to the manufacturer's protocol, without any amplification step, but with an additional size selection step after adapter ligation to remove smaller fragments. The library was quantified by qPCR by using KAPA library quantification kit (Roche Diagnostics Corporation) and a QuantStudio 3 (Thermo Fisher Scientific Inc.), and then sequenced using a HiSeq3000 system (Illumina Inc) with a read setup of 2x151 bp.
Me220
Manufactured by Covaris
Sourced in United States
The ME220 is a high-performance laboratory instrument designed for sample preparation. It utilizes Covaris' proprietary Adaptive Focused Acoustics (AFA) technology to provide precise and controlled sample processing.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 3000 system, by Illumina (2 mentions)
Quantstudio 3, by Thermo Fisher Scientific (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Kapa hyper prep kit, by Roche (2 mentions)
Fragment analyzer, by Agilent Technologies (2 mentions)
Hyper library preparation kit, by Roche (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Accel ngso 2s plus dna doc a160140 v00, by Integrated DNA Technologies (2 mentions)
Formaldehyde, by Merck Group (2 mentions)
Glycine, by Merck Group (2 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Bcl2fastq v2.20 conversion software, by Illumina (1 mentions)
Xgen lockdown probe panel, by Integrated DNA Technologies (1 mentions)
Nextseq 550, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ab1220, by Abcam (1 mentions)
Ab32356, by Abcam (1 mentions)
30 protocols using me220
1
Illumina Library Preparation Protocol
2
Shotgun Sequencing of Genomic DNA
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Library contraction and sequencing were carried out at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign (UIUC). The shotgun genomic DNA libraries were constructed from 200 ng of DNA after sonication with a Covaris ME220 (Covaris) to an average fragment size of 300 bp with the Hyper Library Preparation Kit from Kapa Biosystems (Roche). To prevent index switching, the libraries were constructed using unique dual indexed adaptors from Illumina. Individually barcoded libraries were amplified with 5 cycles of PCR and run on a Fragment Analyzer (Agilent) to confirm the absence of free primers and primer dimers and to confirm the presence of DNA of the expected size range. Libraries were pooled in equimolar concentration and size selected on a 2% agarose gel for fragments 220 bp to 280 bp in length. The pool was further quantitated by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc.). The pooled barcoded shotgun libraries were loaded on a NovaSeq lane for cluster formation and sequencing at a length of 150 nt from each side of the DNA fragments. The fastq read files were generated and demultiplexed with the bcl2fastq v2.20 Conversion Software (Illumina).
3
Automated Targeted NGS Workflow
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All NGS sequencing was performed according to the UroAmplitude procedure, a Clinical Laboratory Improvement Amendments (CLIA) validated diagnostic test. Purified tumor and urine DNA was fragmented by sonication (Covaris ME220, Covaris LLC, Woburn, MA, USA) before undergoing library preparation using the KAPA Hyper Prep Kit (Roche, Basel, Switzerland) protocol and is optimized for automation by a Microlab STARLET (Hamilton). xGen Dual Index adapters are synthesized by IDT and a custom xGen Lockdown Probe panel (Integrated DNA Technology, Coralville, IA, USA) is used for hybridization capture of DNA libraries. The hybridization capture workflow has been optimized for performance on a Nimbus microLAB (Hamilton). Target-enriched libraries are analyzed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and diluted for sequencing. Libraries are loaded into a NextSeq 500/550 High Output Reagent Cartridge (v2, 300 cycles) and sequenced on a NextSeq 550 (Illumina, San Diego, CA, USA).
4
DNA Extraction and Sequencing Protocol
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DNA was extracted for sequencing using the Qiagen Blood and Tissue Kit (Cat. No. 69506). Prior to library preparation 2500 ng of gDNA were sheared with Covaris ME220 (Covaris, Inc.) to a mean fragment size of 550 bp. Library preparation was performed using the VAHTS Universal DNA Library Prep Kit for Illumina (Vazyme Biotech Co.; Ltd) according to the manufacturer's protocol, without any amplification step, but with an additional size selection step after adapter ligation to remove smaller fragments. The library was quantified by qPCR by using KAPA library quantification kit (Roche Diagnostics Corporation) and a QuantStudio 3 (Thermo Fisher Scientific Inc.), and then sequenced using a HiSeq3000 system (Illumina Inc) with a read setup of 2 × 151 bp.
5
DNA Shearing and Sequencing Library Prep
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DNA for each sample was sheared to ~ 300 bp using Covaris ME220 (Covaris, Woburn, MA, USA). The shotgun sequencing library was constructed using the KAPA Hyper Prep Kit (Roche). Libraries were sequenced on DNBSEQ-G400 (BGI, Shenzhen, China) at a 150-bp paired-end.
6
ChIP-qPCR for IRF4 Targets
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Chromatin preparation was performed as previously described (66 (link)). Cells (1 × 107 per condition) were lysed in sonication buffer at a density of 3 × 106 cells/130 μL. Lysate was washed twice with sonication buffer and sonicated for 12 minutes in a Covaris ME220 focused-ultrasonicator using AFA microTUBE-130. IRF4 (4964) and normal rabbit IgG (2729) antibodies were obtained from Cell Signaling Technology. Details of qPCR primers for SUB1, TNFRSF17, and PRDM1, identified as targets for IRF4 by Shaffer et al. (24 (link)), and myoglobin B used as a control, are provided in Supplemental Methods. Percentage input was calculated by linearization of ΔCt (CtIP–Ct1%Input).
7
ChIP-qPCR Assay for Epigenetic Markers
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The ChIP assay was used to measure occupancy of H3K4me2, H3K9me2, and LSD1 proteins (Kyzar et al., 2017 (link), 2019 (link); Bohnsack et al., 2019 (link)). Amygdala tissue was fixed in methanol-free formaldehyde, and chromatin was then sheared to 200- to 500-bp fragments by sonication with the Covaris ME220 (Covaris). The chromatin complex was immunoprecipitated with specific antibodies to H3K9me2 (Abcam ab1220, RRID: AB_449854 , 3 μg/sample), LSD1 (Abcam ab17721, RRID: AB_443964 , 3 μg/sample), and H3K4me2 (Abcam ab32356, RRID: AB_732924 , 3 μg/sample). The precipitated DNA was quantified by qPCR using a CFX Connect qPCR system with iQ SYBR SuperMix (Bio-Rad) and primers designed to specific genomic regions (Table 1 ). Rabbit IgG (Millipore; catalog #NI01; 3 μg/sample) was used as a negative control antibody but did not amplify any genomic region tested in qPCR. Input DNA Ct value was subtracted from the Ct value of each respective sample. The 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)) was used to determine the fold change compared to AIS control groups.
8
Chromatin Immunoprecipitation Sequencing Protocol
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ChIP was performed as previously described6 (link). Samples were sonicated using the Covaris ME220 sonicator (Covaris, Woburn, MA, USA); peak power 75, duty factor 26.66%, 500 cycles/burst for 7.5 minutes. Antibodies directed against hemagglutinin (HA, Abcam Ab9110; RRID: AB_307019), H3K27ac (Abcam, Ab4729, RRID: AB_2118291), and MED1 (Bethyl Laboratories, A300–793A; RRID: AB_577241) were used for immunoprecipitation. For ChIP-seq, the ChIP reactions were scaled to obtain 10 ng DNA. After decrosslinking, the DNA that had undergone ChIP was prepared for sequencing according to the manufacturer’s protocol (Illumina). ChIP-seq was performed on two independent experiments.
9
ChIP-seq protocol with Drosophila normalization
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The cells (1 × 107–8) were fixed and sonicated using a Covaris ME220 according to the manufacturer's recommendations. Ab:chromatin complexes were isolated using magnetic Protein A and Protein G Dynabeads (Invitrogen) and washed three times with 50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% Na deoxycholate. Antibodies used are in Supplemental Table S1 . Samples were washed with Tris-EDTA, eluted, and treated with RNase A and Proteinase K. DNA was purified with a Qiagen PCR purification kit. Samples were analyzed by ChIP qPCR or ChIP-sequencing. Reference normalization (Orlando et al. 2014 (link)) was performed by adding fixed Drosophila melanogaster S2 cells before sonication in a 1:5 ratio. Primers used for qPCR analysis are listed in Supplemental Table S2 . Libraries were generated using Ultra II DNA library preparation kit for Illumina (NEB) and sequenced using a NextSeq 500 (Illumina).
10
Titin Identification via SDS-PAGE and Mass Spectrometry
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The presence of titin in the sample was confirmed by SDS-PAGE (Supplementary Figure S1 ) and mass spectrometry analysis (Supplementary Table S1 ). SDS-PAGE of titin was performed as in [71 (link)] with our modification. In particular, our experiments used a separating gel containing 6.5–7% polyacrylamide prepared as described in [71 (link)]. For shotgun mass spectrometry analysis the sample was solubilized into a buffer (4% sodium dodecyl sulfate in 0.1 M Tris-HCl pH 7.6, 0.1 M dithiothreitol) and incubated for 5 min at 95 °C as described [72 ]. The samples were sonicated (4 × 30 s at 20 W; ME220, Covaris, Woburn, MA, USA), centrifuged (5 min, 16,000× g), and the supernatant was collected. The YM-30 filter (Millipore, Ireland) was used for alkylation and trypsinolysis (14 h, 2 μg of trypsin (Tripsin Gold, Promega, Madison, WI, USA)) according to the FASP method [73 ]. Peptides were desalted using C18 microcolumns and subjected to HPLC-MS/MS analysis using the HPLC Ultimate 3000 RSLCnano system (Thermo Scientific, Waltham, MA, USA) as described [72 ]. For a list of detected titin peptides, see Supplementary Table S1 .
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