Genomic DNA from IS and ID hippocampi was isolated using an AllPrep DNA/RNA Mini Kit (Qiagen). WGBS was performed using a previously published protocol [33 (link)]. Briefly, 1 µg of genomic DNA was fragmented into ~300 bp fragments using a M220 Covaris Ultrasonicator (Covaris, Woburn, MA, USA). Sequencing libraries were generated using a NEBNext genomic sequencing kit (New England Biolabs, Ipswich, MA, USA) and ligated with Illumina methylated paired end adaptors. Libraries were bisulfite-converted using an Imprint DNA modification kit (MilliporeSigma, St. Louis, MO, USA), and the size of 300–600 bp was selected using the Pippin Prep DNA size selection system (Sage Science, Beverly, MA, USA). Libraries were then amplified using Pfu-Turbo Cx Hotstart DNA polymerase (Agilent Technologies, Santa Clara, CA, USA). Paired-end libraries were sequenced to 100 bp on an Illumina hiSeq2000. Three biological replicates for each group were performed in WGBS. WGBS data are available on the Gene Expression Omnibus under GSE98064.
M220 ultrasonicator
Manufactured by Covaris
Sourced in United States
The M220 ultrasonicator is a laboratory instrument designed for sample preparation. It uses high-frequency sound waves to disrupt and homogenize samples, such as tissues or cells, in a controlled and efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
Miseq sequencer, by Illumina (7 mentions)
Truseq nano dna lt library prep kit, by Illumina (5 mentions)
Kapa library quantification kit, by Roche (4 mentions)
Truseq dna sample prep kit, by Illumina (3 mentions)
Pipeline version 1, by Illumina (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Ovation rna seq system, by Tecan (3 mentions)
Bioanalyzer, by Agilent Technologies (3 mentions)
Hiseq 2000, by Illumina (2 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (2 mentions)
Ribolock rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Hiseq x ten, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Qubit dsdna hs assay kit, by Agilent Technologies (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Agilent dna high sensitivity chip, by Agilent Technologies (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Miseq reagent kit, by Illumina (2 mentions)
62 protocols using m220 ultrasonicator
1
Whole Genome Bisulfite Sequencing Protocol
2
Genome Survey Sequencing of Plant Samples
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The fresh leaves of in vivo mother plant and in vitro plantlets were used for genome survey sequencing, respectively. Briefly, genomic DNA was isolated using the CTAB (Cetyl Trimethyl Ammonium Bromide) method [65 (link)]. DNA purity and integrity were monitored on 1% agarose gel electrophoresis, and DNA concentration was measured using Qubit DNA Assay Kit in Qubit 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA). The genomic DNA of each sample was randomly sheared into short fragments of around 350 bp by M220 Covaris ultrasonicator (Covaris, Woburn, MA, USA). The fragments were subjected to library construction using the Truseq Nano DNA HT Sample Preparation Kit (Illumina, San Diego, CA, USA). The paired-end libraries were sequenced using the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA) with read length of 2 × 150 bp by Novogene Co., Ltd. Two sets of raw sequencing reads are available in the Short Read Archive (SRA) database under the accession number of SRR12532532 (the in vitro grown plantlet) and SRR12532533 (the in vivo mother plant).
3
Crosslinking and Chromatin Shearing for ChIRP Analysis
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Five millions of HEK293 cells were crosslinked in 1% methanol free formaldehyde (Thermo Fisher Scientific) for 10 min and then quenched with 0.125 mM glycine for 5 min. Samples were then lysed using the ChIRP lysis buffer (50 mM Tris–HCl pH 7.0, 10 mM EDTA, 1% SDS) supplemented with protease inhibitor cocktail 100× (Thermo Fisher Scientific) and Ribolock RNase inhibitor (Thermo Fisher Scientific). Samples were then sonicated using the Covaris M220 ultrasonicator and 25 μg of sheared chromatin was treated with 200 μg of proteinase K for 45 min at 50°C. DNA was then extracted using GeneJET Gel Extraction kit (Thermo Fisher Scientific) and quantified by the nanodrop 2000. 600 ng of the subsequent DNA were loaded on agarose gel 1.2% to verify the shearing efficiency (Supplementary Figure S1B ). The sheared chromatin was then flash frozen in liquid nitrogen and stored at –80°C for later use.
4
Whole-Exome Sequencing of Familial Patients
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We conducted whole‐exome sequencing (WES) as described (Wang et al., 2014 ). Briefly, genomic DNA was extracted from 2 ml of peripheral blood samples of the three patients and their parents using TIANamp Genomic DNA Kits (TIANGEN, Beijing, China). We sheared 3 μg of DNA from patient II‐3 into lengths of 150–200 bp using a Covaris® M220 Ultrasonicator (Covaris Inc.). An adapter‐ligated library was generated using an Agilent SureSelect Target Enrichment System (Agilent Technologies, Inc.), and a capture library including both coding exons and flanking intronic regions was produced using SureSelect XT Human All Exon V6 reagent kits (Agilent Technologies Inc.). Clusters were then generated by isothermal bridge amplification using an Illumina cBot station, and sequenced using an Illumina X10 System (Illumina Inc.).
The sequence reads were aligned to a reference human genome (GRCh37/hg19) using NextGENe® software (SoftGenetics LLC). All single nucleotide variants (SNV) and indels were uploaded in VCF format for Ingenuity® Variant Analysis™ (Ingenuity Systems), bioinformatics analysis and interpretation.
The sequence reads were aligned to a reference human genome (GRCh37/hg19) using NextGENe® software (SoftGenetics LLC). All single nucleotide variants (SNV) and indels were uploaded in VCF format for Ingenuity® Variant Analysis™ (Ingenuity Systems), bioinformatics analysis and interpretation.
5
Whole-Genome Bisulfite Sequencing Protocol
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DNA was extracted with DNeasy Blood and Tissue Kit (Qiagen; Valencia, CA, USA), and each DNA sample was spiked with 1% unmethylated lambda DNA (Promega; Madison, WI, USA) to evaluate the bisulfite conversion efficiency. The genomic DNA (500 ng) was fragmented with Covaris M220 ultrasonicator (Covaris; Woburn, MA, USA) to an average size of 350 bp. End repair and methylated adaptor ligation was conducted with NEBNext Ultra End Repair/dA-Tailing Module, Ligation Module and NEBNext Multiplex Oligos for Illumina (Methylated Adaptor, Index Primers Set 1; New England Biolabs; Ipswich, MA, USA). DNA fragments between 400 and 500 bp were selected for library construction with Ampure XP beads (Beckman Coulter; Brea, CA, USA). Bisulfite conversion was performed on samples using the EZ DNA Methylation kit (Zymo Research; Irvine, CA, USA) with modified single-stranded DNA fragments amplified using the Kapa HiFi U+ HotStart ReadyMix (Kapa Biosystems; Wilmington, MA, USA) with primers (NEBNext Multiplex Oligos for Illumina). A final size selection was performed to enrich the library for a range between 300 and 500 bp. Constructed libraries were evaluated on the Agilent 2100 Bioanalyzer (Agilent Technologies; Santa Clara, CA, USA) and then sequenced on the Illumina HiSeq X Ten (Illumina; San Diego, CA, USA) using the 150-bp paired-end mode.
6
Paired-end Metagenomic Sequencing and Assembly
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For each sample, 1 μg of genomic DNA was sheared into fragments (approximately 450 bp in length) using the Covaris M220 ultrasonicator (Covaris Inc., Woburn, MA). Paired-end metagenomic libraries were constructed using the TruSeq DNA Sample Prep Kit (Illumina, San Diego, CA) and sequenced on the Illumina HiSeq X Ten platform.
After sequencing, the raw data were filtered to remove adaptor contaminations and low-quality reads using Trimmomatic (Bolger et al., 2014) (link). The reads were further screened with the BWA-mem algorithm (Li, 2013) to remove host-genome pollution by mapping to Bos taurus ARS-UCD1.2, which was obtained from the National Center for Biotechnology Information (Bethesda, MD). Subsequently, the remaining clean reads were assembled into contigs using MEGAHIT (Li et al., 2015) (link) with a parameter of "-min-contig-len 500." Gene predictions were conducted using Prodigal (Hyatt et al., 2010) (link), and all the open reading frames generated by the samples were clustered into a non-redundant gene set using the Cluster Database at High Identity with Tolerance program (Fu et al., 2012) (link) with a sequence identity cut-off of 0.95. The sequencing data for our samples have been deposited into Sequence Read Archive under accession number PRJNA639405.
After sequencing, the raw data were filtered to remove adaptor contaminations and low-quality reads using Trimmomatic (Bolger et al., 2014) (link). The reads were further screened with the BWA-mem algorithm (Li, 2013) to remove host-genome pollution by mapping to Bos taurus ARS-UCD1.2, which was obtained from the National Center for Biotechnology Information (Bethesda, MD). Subsequently, the remaining clean reads were assembled into contigs using MEGAHIT (Li et al., 2015) (link) with a parameter of "-min-contig-len 500." Gene predictions were conducted using Prodigal (Hyatt et al., 2010) (link), and all the open reading frames generated by the samples were clustered into a non-redundant gene set using the Cluster Database at High Identity with Tolerance program (Fu et al., 2012) (link) with a sequence identity cut-off of 0.95. The sequencing data for our samples have been deposited into Sequence Read Archive under accession number PRJNA639405.
7
Illumina MiSeq Sequencing of Sheared DNA
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DNA extractions were performed as described above. DNA was submitted to the University of Wisconsin-Madison Biotechnology Center. DNA concentration was verified using the Qubit dsDNA HS Assay Kit (Life Technologies, Grand Island, NY). Samples were prepared according the TruSeq Nano DNA LT Library Prep Kit (Illumina Inc., San Diego, California, USA) with minor modifications. A maximum of 200 ng of each sample was sheared using a Covaris M220 Ultrasonicator (Covaris Inc, Woburn, MA, USA). Sheared samples were size selected for an average insert size of 550 bp using Spri bead based size exclusion. Quality and quantity of the finished libraries were assessed using an Agilent DNA High Sensitivity chip (Agilent Technologies, Santa Clara, CA) and Qubit dsDNA HS Assay Kit, respectively. Libraries were standardized to 2 μM. Paired-end, 150 bp sequencing was performed using v2 SBS chemistry on an Illumina MiSeq sequencer. Images were analyzed using the Illumina Pipeline, version 1.8.2.
8
Comprehensive NGS Profiling of Lung Cancer
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In total, 12,533 samples were analysed using the DNA‐based NGS method. Library preparation for 68 lung cancer‐related genes, covering 0.345 Mb of the human genome, was performed using a DNA capture‐based NGS panel (Burning Rock Biotech, Guangzhou, PR China) (supplementary material, Table S1 ). A total of 100–200 ng of genomic DNA from FFPE tissues was fragmented using Covaris M220 ultrasonicator (Covaris Woburn, MA, USA). An enriched library was then used to hybridise with the capture probes. The amount and size of the library were assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed using a NextSeq 550 sequencer (Illumina, Inc., San Diego, CA, USA). NGS sequence reads were aligned to the hg19 version of the human genome using BWA‐MEM 0.7.10. The data processing methods employed here have been described previously [11 (link)].
9
Whole Genome Sequencing of Streptococcus pneumoniae
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Genomic DNA preparation, library construction, whole genome sequencing (WGS), and bioinformatics pipeline features have been previously described (Li et al., 2016 (link), 2017 (link); Metcalf et al., 2016a (link),b (link)). Streptococcus pneumoniae strains were cultured on Trypticase soy agar supplemented with 5% sheep blood and incubated overnight at 37°C in 5% CO2. Genomic DNA for short-read WGS was extracted manually using a modified QIAamp DNA mini kit protocol (Qiagen, Inc., Valencia, CA, USA). Nucleic acid concentration was quantified by an Invitrogen Qubit assay (Thermo Fisher Scientific Inc., Waltham, MA, USA) and samples were sheared using a Covaris M220 ultrasonicator (Covaris, Inc., Woburn, MA, USA) programmed to generate 500-bp fragments. Libraries were constructed on the SciCloneG3 (PerkinElmer Inc., Waltham, MA, USA) using a TruSeq DNA PCR-Free HT library preparation kit with 96 dual indices (Illumina Inc., San Diego, CA, USA) and quantified by a KAPA qPCR library quantification method (Kapa Biosystems Inc., Wilmington, MA, USA). WGS was generated employing two MiSeq instruments and the MiSeq v2 500 cycle kit (Illumina Inc).
10
High-Throughput Genomic DNA Sequencing
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Genomic DNAs were prepared with a genomic DNA purification kit (Wizard; Promega), and DNA concentrations were determined using the Qubit dsDNA high-sensitivity assay kit (Life Technologies). Sequencing was performed at the University of Wisconsin—Madison Biotechnology Center. Samples were prepared according to the TruSeq Nano DNA LT library prep kit (Illumina Inc.). Briefly, samples were sheared using a Covaris M220 ultrasonicator (Covaris Inc.) and size selected for an average insert size of 550 bp using solid-phase reversible-immobilization bead-based size exclusion. The quality and quantity of the finished libraries were analyzed using an Agilent DNA1000 chip and a Qubit dsDNA high-sensitivity assay kit. Libraries were standardized to 2 nM. Paired-end, 250-bp sequencing was performed using the Illumina MiSeq sequencer and a MiSeq 500-bp (v2) sequencing cartridge. Images were analyzed using the standard Illumina pipeline (version 1.8.2).
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