Tag: Ampure XP

Comparison of Oxford Nanopore Technologies and Illumina MiSeq sequencing with mock communities and agricultural soil

Study sites Soils were collected from two different sites (ARDEC: Colorado State University’s Agricultural Research, Development and Education Center in Fort Collins, CO; and CPCRC: USDA Columbia Plateau Conservation Research Center in Pendleton, OR). At each site, four replicate plots of no-till corn (ARDEC) or no-till annual wheat (CPCRC) were…

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acCRISPR: an activity-correction method for improving the accuracy of CRISPR screens

acCRISPR framework acCRISPR performs essential gene identification by calculating two scores for each sgRNA, namely the cutting score (CS) and the fitness score (FS). CS and FS are the log2-fold change of sgRNA abundance in the appropriate treatment sample with respect to that in the corresponding control sample (see Supplementary…

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Epigenetic dysregulation from chromosomal transit in micronuclei

Cell culture Cell lines (MDA-MB-231, 4T1 and RPE-1) were purchased from the American Type Culture Collection (ATCC). TP53-knockout MCF10A, TP53-knockout RPE-1 and Trex1 knockout 4T1 cells were gifts from the Maciejowski laboratory at the Memorial Sloan Kettering Cancer Center (MSKCC). OVCAR-3 cells were a gift from J. D. Gonzales. All…

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Heritable transcriptional defects from aberrations of nuclear architecture

Cell culture and cell line construction Cells were cultured at 37 °C in 5% CO2 atmosphere with 100% humidity. Telomerase-immortalized RPE-1 retinal pigment epithelium cells (CRL-4000, American Type Culture Collection), U2OS osteosarcoma cells (HTB-96, American Type Culture Collection) and derivative cell lines were grown in DMEM/F12 (1:1) medium without phenol red…

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Characterization of nuclear DNA diversity in an individual Leymus chinensis

Introduction The definition of an individual organism, traditionally defined by the invariable presence of a physiological unit and genetic homogeneity, is challenged by situations such as intraorganismal genetic heterogeneity (IGH), which refers to the presence of more than one genotype in a single organism (Schweinsberg et al., 2015). Cells within a…

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Activation-inducible CAR expression enables precise control over engineered CAR T cell function

Primary cells PBMCs were either isolated from fresh buffy coats via density gradient centrifugation using Biocoll (Biochrom) or selected from a Leukapheresis material using in house T cell selection technology ATC. Buffy coats were obtained from autologous adult female or male blood donors at the Institute for Anesthesiology, German heart…

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An integrated tumor, immune and microbiome atlas of colon cancer

Samples used in this observational cohort study (tumor tissue and matched healthy colon tissue, AC-ICAM cohort) are from patients with colon cancer diagnosed at Leiden University Medical Center, the Netherlands, from 2001 to 2015 that did not object for future use of human tissues for scientific research and that were…

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Methanol fixation is the method of choice for droplet-based single-cell transcriptomics of neural cells

hiPSC cell culture and differentiation hiPSCs were maintained on 1:40 matrigel (Corning, #354277) coated dishes in supplemented mTeSR-1 medium (StemCell Technologies, #85850) with 500 U ml−1 penicillin and 500 mg ml−1 streptomycin (Gibco, #15140122). For the differentiation of cortical neurons the protocol described previously21 was followed with slight modifications. Briefly, hiPSC colonies were seeded…

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Gut microbiome profiling of neonates using Nanopore MinION and Illumina MiSeq sequencing

Introduction Perturbations in the infant gut microbiome during early life affect growth, development, and long-term health (Sherman, 2010; Carl et al., 2014; Tarr and Warner, 2016). Preterm infants have a physiologically and anatomically immature gastrointestinal tract that is known to be more permeable than that of term infants, and their…

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In-depth Temporal Transcriptome Profiling of Monkeypox and Host Cells using Nanopore Sequencing

Figure 1 shows the detailed workflow of the study. Fig. 1 General overview of the study. Briefly, MPXV was isolated from a skin lesion and then was used to infect CV-1 cells. After the designated infection times, total RNA was isolated and sequenced using direct cDNA sequencing protocol on ONT’s MinION platform….

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Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol | PLOS ONE – Molecular Biology Protocol – Restriction Digest of Plasmid …

Abstract A waxing variety of “genotype-by-sequencing” (GBS) methods use restriction enzymes and high throughput DNA ordering to generation data for a set of genomic loci, allowing the simultaneous discovery press genotyping of thousands of polymorphisms in a set of multiplexed samples. We evaluated a “double-digest” restriction-site associated DNA sequencing (ddRAD-seq)…

Continue Reading Amplification Biases and Consistent Recovery of Loci in a Double-Digest RAD-seq Protocol | PLOS ONE – Molecular Biology Protocol – Restriction Digest of Plasmid …

Roles of adenine methylation in the physiology of Lacticaseibacillus paracasei

Bacterial strains and cultivation Twenty-eight L. paracasei isolates (including L. paracasei Zhang) and a pglX gene-inactivated strain of L. paracasei Zhang were obtained from the Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, at the Inner Mongolia Agricultural University of China. For strain activation, the bacteria were cultivated…

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Illumina TruSeq Stranded total Sample Preparation Protocol | CHMI services – TruSeq Stranded Total RNA

Preparation of total transcriptome libraries for sequencing on an Illumina dais Edit me Documentation TruSeq Running Absolute RNA Sample Prep Guide. This method makes a cDNA library of all RNA molecules presentational in your spot after rRNA depletion. Important: the succeed of this kit is dependent on of ability of…

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Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

Ethics statement All experiments in this study comply with guidelines of the U.S. Public Health Service and were approved by the Institutional Biosafety Committee at the University of Pittsburgh. All animal husbandry and experimental procedures were conducted in accordance with U.S. Public Health Service policy and approved by the Institutional…

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A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants | Plant Methods

Plant material Streptocarpus grandis (RBGE lineage 19771210) and Streptocarpus kentaniensis (RBGE lineage 19951992) were cultivated in the glasshouses at the Royal Botanic Garden Edinburgh. DNA extraction method for Streptocarpus PacBio HiFi long-read sequencing To develop the protocol, small scale extractions were carried out with approx. 3 g leaf tissue as input…

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RNA-DNA interactomes of three prokaryotes uncovered by proximity ligation

Cell strains E. coli DH5α and B. subtilis 168 strains were grown overnight in Luria-Bertani broth at 37 °C and 180 rpm to a final OD600 ~1.7. B. subtilis strain 168 was kindly provided by Dr. S.A. Dubiley (Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia). T. adornatum strain…

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Gut taste receptor type 1 member 3 is an intrinsic regulator of Western diet-induced intestinal inflammation | BMC Medicine

Study design The objective of the study was to evaluate the influence of long-term WD intake on intestinal inflammation and investigate possible mechanisms by which WD intake could affect IBD development. To this end, mice were fed normal diet (ND) or WD for 10 weeks, and bowel inflammation was evaluated through…

Continue Reading Gut taste receptor type 1 member 3 is an intrinsic regulator of Western diet-induced intestinal inflammation | BMC Medicine

Construction of high coverage whole-genome sequencing libraries from single colon crypts without DNA extraction or whole-genome amplification | BMC Research Notes

Tissue collection and crypt isolation A small piece of colon is collected from individuals who have undergone surgery to remove part of the colon under the standard of care at either Keck Hospital of USC or Children’s Hospital Los Angeles through the Norris Comprehensive Cancer Center Translational Pathology Core. A…

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Single duplex DNA sequencing with CODEC detects mutations with high sensitivity

Ethical approval, DNA samples and oligonucleotides All patients provided written informed consent to allow the collection of blood and/or tumor tissue and the analysis of clinical and genetic data for research purposes. The IRB of the Dana-Farber Cancer Institute and New York University Grossman School of Medicine approved these protocols….

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MYD88L265P and MYD88other variants show different molecular characteristics and prognostic significance in diffuse large B-cell lymphoma

Patient selection and data collection This study retrospectively collected 475 patients diagnosed with de novo DLBCL by two experienced pathologists at the CHCAMS and Suzhou Municipal Hospital from February 6th, 2007 to May 20th, 2022. A total of 132 patients with MYD88 variations were included for analysis. The inclusion criteria…

Continue Reading MYD88L265P and MYD88other variants show different molecular characteristics and prognostic significance in diffuse large B-cell lymphoma

SituSeq: an offline protocol for rapid and remote Nanopore 16S rRNA amplicon sequence analysis

For the initial test of this protocol, DNA extraction, PCR, Nanopore library preparation, Nanopore sequencing, and subsequent data analysis were conducted at sea aboard the R/V Atlantic Condor in August 2021 [26]. This investigational effort resulted in the sequencing and analysis of deep sea sediment samples within hours of their…

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Uropathogenic Escherichia coli infection-induced epithelial trained immunity impacts urinary tract disease outcome

Ethics statement All animal experimentation was conducted according to the National Institutes of Health guidelines for the housing and care of laboratory animals. All experiments were performed in accordance with institutional regulations after review and approval by the Animal Studies Committee at Washington University School of Medicine in St Louis,…

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Toxins | Free Full-Text | Metagenome Analysis Identifies Microbial Shifts upon Deoxynivalenol Exposure and Post-Exposure Recovery in the Mouse Gut

1. Introduction Fusarium fungi are world-wide producers of a range of mycotoxins. Deoxynivalenol (DON) belonging to the group B trichothecenes, is one of the most prevalent food-associated mycotoxins mainly produced by Fusarium graminearum and Fusarium culmorum, and frequently contaminates cereals and cereal products [1,2]. Almost half of a total of…

Continue Reading Toxins | Free Full-Text | Metagenome Analysis Identifies Microbial Shifts upon Deoxynivalenol Exposure and Post-Exposure Recovery in the Mouse Gut

Whole-genome doubling drives oncogenic loss of chromatin segregation

Cell culture hTERT-RPE-1 WT and hTERT RPE-1 TP53−/− (46, XX)27 cells were a gift from J. Korbel. The cells were grown in DMEM/F-12, GlutaMAX (10565018) supplemented with 10% FBS (Thermo Fisher Scientific, 10270106) and 1% antibiotic–antimycotic (Thermo Fisher Scientific, 15240062). CP-A (KR-42421) (47, XY) cells were purchased from the American Type…

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Determining the most accurate 16S rRNA hypervariable region for taxonomic identification from respiratory samples

This prospective observational study (NCT04803695) complied with the Declaration of Helsinki (current version, Fortaleza, Brazil, October 2013). Our institution’s Internal Review Board approved the study, and all patients gave their written informed consent (No. HCB/2018/0236, Hospital Clinic Barcelona). Sputum samples and DNA extraction Thirty-three sputum samples were collected from patients…

Continue Reading Determining the most accurate 16S rRNA hypervariable region for taxonomic identification from respiratory samples

A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations

Viral RNA samples Initial development and validation of the assays was performed using four in vitro cultured viruses maintained at LSHTM, for which stocks were obtained from the National Institute for Biological Standards and Controls (NIBSC): 19A SARS-CoV-2 (BetaCoV/Australia/VIC01/2020, CVA-GLA-1, CVA-GLA-2, CVA-GLA-3); two in vitro cultured viruses obtained from BEI…

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Hypoxia-induced transcriptional differences in African and Asian versus European diabetic cybrids

Human subjects Research involving human subjects was approved by the Institutional Review Board of the University of California, Irvine (#2003-3131). All enrolled patients provided written, informed consent. Clinical investigations were performed based on the ethical principles of the Declaration of Helsinki26. Cybrid generation Patient blood was collected in tubes containing…

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Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils

1. Introduction Soil harbors high abundance and diversity of prokaryotic microorganisms, with 1 g of soil containing up to 10 billion microbial cells (Torsvik and Øvreås, 2002) and 103–106 phylotypes (Bickel and Or, 2020), including many microorganisms that play critical biogeochemical roles (Crowther et al., 2019). With continuous expansion of prokaryotic…

Continue Reading Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils

BdLT-Seq as a barcode decay-based method to unravel lineage-linked transcriptome plasticity

Cell lines Immortalised HA1E (hTERT and SV40ER) and tumourigenic HA1ER cells (hTERT, SV40ER and HRAS-G12V) from stepwise tumourigenesis models generated from normal human embryonic kidney cells were obtained from Dr. Hahn (Broad Institute of MIT and Harvard, Cambridge, USA). Cells were cultured in DMEM (1 g/L glucose) (Gibco, cat. #10567014) supplemented…

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QIAseq FX DNA Library Kit Protocol – QIAseq FX DNA Library Kit Protocol Procedure: 1. Acquire high

QIAseq FX DNA Library Kit Protocol Procedure: 1. Acquire high quality gDNAsamples. 2. Set up a thermocycler to incubate for 30 minutes at 32℃, with the heated lid at 70℃. Run the program and pause until the block reaches 4℃. 3. In a tube on ice, prepare and mix the…

Continue Reading QIAseq FX DNA Library Kit Protocol – QIAseq FX DNA Library Kit Protocol Procedure: 1. Acquire high

MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming

Cell culture Normal Human Melanocytes (NHM) were cultured in Dermal Cell Basal Medium (ATCC) with the addition of 5 µg/ml Insulin, 50 µg/ml Ascorbic Acid, 6 mM L-Glutamine, 1.0 µM Epinephrine, 1.5 mM Calcium Chloride, Peptide Growth Factor and M8 Supplement. Dermal fibroblasts (DFs) were isolated from neonatal mice and iPS reprogramming was performed as…

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Dryad | Data — MinION sequencing data of mtDNA from BH10 cells

Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic because of its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required…

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Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains

This research complies with all relevant ethical regulations approved by the Human Investigation Review Board of Albert Szent-Györgyi Clinical Centre of the University of Szeged and the National Biodiversity Authority (NBA) of India. Permission for the faecal sample collection was obtained from the Human Investigation Review Board of Albert Szent-Györgyi…

Continue Reading Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains

Transcriptional reprogramming of skeletal muscle stem cells by the niche environment

Isolation of pure populations of MuSCs and niche cells by fluorescence-activated cell sorting (FACS) Hindlimb muscles were dissected from young (4–6 weeks old) or aged (22–26 months old) C57BL/6J (Jackson Laboratory, 000664) mice and minced until no visible tissue chunks were visible. Muscle was then digested in a 15 ml Falcon…

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Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo

Materials Medakamo hakoo 311 was obtained from the personal aquarium of Prof. Kuroiwa (Kagurazaka, Tokyo, Japan)4. The M. hakoo strain was cultured in 0.05% HYPONeX (HYPONeX Japan Corp., Ltd., Osaka, Japan) liquid medium and on 0.05% HYPONeX gellan gum-based solid medium in plates. Cyanidioschyzon merolae 10D (Toda et al. 1995)…

Continue Reading Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo

The automated Galaxy-SynBioCAD pipeline for synthetic biology design and engineering

Retrosynthesis from target to chassis Typically, the target compound, also named “source compound” is the compound of interest one wishes to produce, while the precursors are usually compounds that are natively present in a chassis strain. In the present implementation, the target can be any chemical that could be described…

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Live-seq enables temporal transcriptomic recording of single cells

Biological materials RAW264.7, 293T and HeLa cells were obtained from ATCC. RAW264.7 cells with Tnf-mCherry reporter and relA-GFP fusion protein (RAW-G9 clone) were kindly provided by I.D.C. Fraser (National Institutes of Health). The IBA cell line derived from the stromal vascular fraction of interscapular brown adipose tissue of young male…

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Pollinator sharing, copollination, and speciation by host shifting among six closely related dioecious fig species

Sampling Six dioecious fig species (F. erecta, F. formosana, F. vaccinioides, F. abelii, F. pyriformis, and F. variolosa) and their pollinator wasp species were examined in this study. As mentioned in the Introduction, these fig species were considered to be well suited for this study because they are distributed in…

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BTG2 gene predicts poor outcome in PT-DLBCL

Introduction Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a rare and aggressive form of mature B-cell lymphoma.1–3 PT-DLBCL was the most common type of testicular tumor in men aged over 60 and characterized by painless uni- or bilateral testicular masses with infrequent constitutional symptoms.4–6 PT-DLBCL shows significant extranodal tropism,…

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Single-cell delineation of lineage and genetic identity in the mouse brain

STICR lentiviral library preparation and validation We synthesized a high-complexity lentivirus barcode library that encodes approximately 60–70 million distinct oligonucleotide RNA sequences (STICR barcodes). STICR barcodes comprised three distinct oligonucleotide fragments cloned sequentially into a multicloning site within the 3′ UTR of an enhanced green fluorescent protein (eGFP) transgene under…

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Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory

The mammalian genome is characterized by widespread methylation of cytosine residues. After fertilization, however, both maternal and paternal genomes undergo extensive demethylation, reaching a low point in the blastocyst (1⇓⇓–4). The embryo genome is then remethylated by the activity of de novo DNA methylation enzymes (5). Mouse embryonic stem (ES)…

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Emergence and expansion of highly infectious spike protein D614G mutant SARS-CoV-2 in central India

COVID-19 laboratory screening Nasopharyngeal/Nasal/Oropharyngeal swabs in viral transport medium (VTM) received from acute phase patients with defined symptoms, asymptomatic cases with contact history with positive patients/ travel history were processed for laboratory confirmation of SARS-CoV-2 at Defence Research and Development Establishment, (DRDE) Gwalior, M.P., India. These samples were referred for…

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PacBio sequencing output increased through uniform and directional fivefold concatenation

Strategy and design of the method We sought to develop a simple method to increase the sequencing capability of PacBio CCS to sequence several diverse DNA libraries ~ 870 bp in length that encoded protein variants originating from a directed evolution campaign. To achieve an increase in the throughput of a PacBio sequencing…

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Multiform antimicrobial resistance from a metabolic mutation

Abstract A critical challenge for microbiology and medicine is how to cure infections by bacteria that survive antibiotic treatment by persistence or tolerance. Seeking mechanisms behind such high survival, we developed a forward-genetic method for efficient isolation of high-survival mutants in any culturable bacterial species. We found that perturbation of…

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