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Tag: Ampure XP
Domestic pigs are susceptible to experimental infection with non-human primate-derived Reston virus without the need for adaptation
Ethics and animal welfare statement All infectious work with RESTV, including sample inactivation, was performed in the Containment Level 4 laboratory (CL4) in accordance with the policies and protocols outlined by the Canadian Science Centre for Human and Animal Health Institutional Biosafety Committee. All animal work was performed in strict…
Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir
Participants and blood collection A total of n = 23 HIV-1 seropositive individuals on stably suppressive ART were included in this study (Supplementary Table 1). Participants were recruited at Ghent University Hospital. 2/23 individuals are female, 21/23 are male; the limited representation of female individuals in our study is a direct reflection of…
Genomic hypomethylation in cell-free DNA predicts responses to checkpoint blockade in lung and breast cancer
Lung cancer ICB cohort Advanced non-small cell lung carcinoma patients who were treated with anti-PD-1/PD-L1 monotherapy at Samsung Medical Center, Seoul, Republic of Korea were enrolled for this study. The present study has been reviewed and approved by the Institutional Review Board (IRB) of the Samsung Medical Center (IRB no….
Benchmarking DNA isolation methods for marine metagenomics
Scheme of the study The overall pipeline of our study is presented in Fig. 1 and is described in detail in the methods section. We processed three types of samples: fresh water, sea sediment, and digestive system of a marine invertebrate M. gigas (“gut flora”). These samples were treated in triplicates…
Thyroid hormone-regulated chromatin landscape and transcriptional sensitivity of the pituitary gland
Mouse genetic models The ThrbHAB allele expresses TRβ proteins (TRβ1 and TRβ2) fused to a peptide with a hemagglutinin (HAx2) tag and a site for biotinylation by prokaryotic BirA ligase, modified from a published tag30. The tag was inserted at the endogenous Thrb gene by homologous recombination in W9.5 (129/Sv)…
Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer
Mice The research conducted in this study complied with all of the relevant ethical regulations. The animal protocols were approved by and performed in accordance with the Institutional Animal Care and Use Committee of St. Jude Children’s Research Hospital. C57BL/6, OT-I50, pmel51 and Rosa26-Cas9 knock-in52 mice were purchased from The…
A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of non-coding elements in vivo
Animal models All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) at Washington University in St. Louis, MO. Veterinary care and housing was provided by the veterinarians and veterinary technicians of Washington University School of Medicine under Dougherty lab’s approved IACUC protocol. All protocols…
Whole genome sequencing in high-grade cervical intraepitheli… : Medicine
1. Introduction Cervical cancer (CC) is the third most common cancer in women worldwide and has a high mortality rate among women. In 2008, CC was responsible for 275,000 deaths, thereby being the fourth leading cause of cancer death in females worldwide.[1,2] In China, CC is the second most…
Recovery of microbial DNA by agar-containing solution from extremely low-biomass specimens including skin
Ethical considerations This study complies with relevant institutional, national, and international guidelines and legislation. The Ethics Committee at the RIKEN Center for Integrative Medical Sciences (H30-4) approved this study, and written informed consent was obtained from all volunteers who provided specimens. Sampling solutions The AgST solution was prepared as follows:…
Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillance
Sample collection and spiking Chicken fecal samples were collected and processed as follows: one spoonful of fecal material (≈ 1 g) was collected and stored in a DNA/RNA Shield™ Fecal Collection Tube R1101 containing 9 ml of DNA/RNA-shield (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. The sample was mixed…
Expression profiles of circRNAs and biomarkers for COPD
Introduction Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease featured by airflow obstruction with high prevalence, morbidity, and mortality. It is reported that COPD is the third leading cause of death globally at present, and the incidence of COPD in adults over 40 years is more than…
Unzipped chromosome-level genomes reveal allopolyploid nematode origin pattern as unreduced gamete hybridization
Nematode materials and species identification Mi, Mj, and Mg were collected from farmlands in Wuhan city of Hubei province, Longyan city of Fujian province, and Changsha city of Hunan Provinces, respectively. Two Ma samples were collected from farmlands in Shenyang city of Liaoning province and Shiping city of Yunnan province….
Comparative analysis of shotgun metagenomics and 16S rDNA sequencing of gut microbiota in migratory seagulls [PeerJ]
Introduction The microbiomes of different host organisms are currently described by using culture-independent sequencing methods (Harvey & Holmes, 2022). Shotgun metagenomic and 16S rDNA sequencing methods are commonly used to identify the taxonomic composition of microbial communities in several studies (de Melo et al., 2020; Mackelprang et al., 2011; Monaco…
Mouse genome rewriting and tailoring of three important disease loci
BAC plasmids Human (CH17-203N23, CH17-449P15 and CH17-339H2) and mouse (RP23-51O13, RP23-75P20 and RP23-204E8) BACs were purchased from BACPAC Resources Center. Yeast–bacterium shuttle vector pLM1050 was modified by L. Mitchell based on a previous study28. pWZ699 was constructed by inserting a cassette containing pPGK-ΔTK-SV40pA transcription unit and the Actb gene into…
Cleanup – NGS Library Prep | Innovators in DNA Sequencing Technologies
Magnetic beads enable cleanup reactions as well as a size selection to recover the desired fragment lengths needed for the specific sequencing application, and are the most common approach in NGS library cleanup, applied both manually and automated. This cost-effective approach is based on inert beads that contain polystyrene…
Invasive Californian death caps develop mushrooms unisexually and bisexually
Mushroom collecting Sporocarps were collected from various herbaria and during three expeditions to Point Reyes National Seashore (PRNS), California in 2004, 2014 and 2015, and in 2015 from three sites in Portugal. A total of 86 sporocarps were collected: 67 Californian sporocarps (one early herbarium sample dates to 1993), 11…
Impact of humid climate on rheumatoid arthritis faecal microbiome and metabolites
Animals and husbandry details Six-week-old healthy male SD rats were used in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. The study is reported in accordance with…
Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment
Culture of hiPS cells The hiPS cell lines used in this study were validated as previously described51. The SEC61B-mEGFP hiPS cell line (AICS-0059 cl.36) was developed at the Allen Institute for Cell Science (www.allencell.org/cell-catalog.html) and is distributed through Coriell43. The CAG::Cas9 cell line was genetically engineered in the laboratory (the…
Comparative analysis of bioinformatics tools to characterize SARS-CoV-2 subgenomic RNAs
Introduction The coronavirus infectious diseases-2019 worldwide pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently ongoing after being emerged from Wuhan (China) in December 2019 (Ecdc, 2020; Zhu et al, 2020; Spiteri et al, 2020). Up to now, SARS-CoV-2 has infected more than 750 million people and…
capTEs enables locus-specific dissection of transcriptional outputs from reference and nonreference transposable elements
Cell culture All cell lines were grown in 6 cm dishes at 37 °C in a 5% CO2 incubator. The K562, MDA-MB-231 and HCT 116 cell lines were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics (pen-strep). NCM460 cells were cultured in RPMI 1640 medium supplemented…
Cell-free chromatin immunoprecipitation to detect molecular pathways in heart transplantation
Abstract Existing monitoring approaches in heart transplantation lack the sensitivity to provide deep molecular assessments to guide management, or require endomyocardial biopsy, an invasive and blind procedure that lacks the precision to reliably obtain biopsy samples from diseased sites. This study examined plasma cell-free DNA chromatin immunoprecipitation sequencing (cfChIP-seq) as…
A subset of viruses thrives following microbial resuscitation during rewetting of a seasonally dry California grassland soil
Field sample collection Topsoil samples (0–15 cm, roughly 0.5 m3) from replicate field plots were collected from the Hopland Research and Extension Center (HREC) in Northern California, which is unceded land of the Shóqowa and Hopland People, on August 28th, 2018 after experiencing mean annual precipitation during the rainy season, see our…
Viruses | Free Full-Text | Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses
1. Introduction Avian orthoreoviruses (avian reoviruses, ARVs) are a persistent challenge to poultry producers in the United States and globally. Infection with ARVs has been associated with a variety of symptoms and syndromes in commercial poultry, including tenosynovitis/viral arthritis, enteric symptoms such as watery diarrhea, respiratory symptoms, myocarditis, viral hepatitis,…
Symmetric inheritance of parental histones governs epigenome maintenance and embryonic stem cell identity
The research in this study was conducted under the ethical approval of the Danish Regulatory Authority under project license 2018-15-0201-01520. Cell culture and differentiation assays WT, MCM2-2A, MCM2-R and POLE4-KO mouse ESCs used in this study were derived from the male, E14JU cell line with a 129/Ola background81. Genome editing…
Gut microbial carbohydrate metabolism contributes to insulin resistance
Study participants and data collection The study participants were recruited from 2014 to 2016 during their annual health check-ups at the University of Tokyo Hospital. The individuals included both male and female Japanese individuals aged from 20 to 75 years. The exclusion criteria were as follows: established diagnosis of diabetes,…
Epitope editing enables targeted immunotherapy of acute myeloid leukaemia
Sleeping Beauty transgene overexpression Bidirectional Sleeping Beauty plasmids encoding the codon-optimized cDNA of the gene of interest (FLT3, KIT, CD123) or their mutated variants were cloned downstream of an EEF1A1 promoter. The same construct included a reporter cassette comprising a fluorescent protein (mCherry or mTagBFP2 for FLT3 and KIT, respectively)…
Selective enrichment of plasma cell-free messenger RNA in cancer-associated extracellular vesicles
Clinical samples and plasma preparation Blood samples from control individuals and patients with multiple myeloma, liver cancer, and lung cancer were obtained from Oregon Health and Science University (OHSU) by Knight Cancer Institute Biolibrary and Oregon Clinical and Translational Research Institute (OCTRI). All samples were collected under OHSU institutional review…
A highly efficient scheme for library preparation from single-stranded DNA
Vector construction The gene encoding VTopoI (GenBank: L13447.1) was synthesized by Eurofins Genomics Inc (Tokyo, Japan), with codon optimization for Escherichia coli K-12, exclusion of internal BamHI and EcoRI sites, and their inclusion at the 5′- and 3′-ends of the DNA, respectively (Supplementary Sequences). The BamHI-EcoRI fragment of the gene…
Mitochondrial impairment and synaptic dysfunction are associated with neurological defects in iPSCs-derived cortical neurons of MERRF patients | Journal of Biomedical Science
Cell culture of MERRF iPSCs The 6 lines of human iPSCs used in this study were generated from the skin fibroblasts established from 3 patients harboring the MERRF syndrome-specific m.8344A > G mutation. Among them, two patients are the members of a Taiwanese MERRF family, a 15-year-old girl (M1-iPSCs) and her 13-year-old…
Transient naive reprogramming corrects hiPS cells functionally and epigenetically
Cell culture All cell lines used and derived by different approaches in this study are listed in Supplementary Table 1. Detailed information about the experimental design, materials and reagents is presented in the Reporting Summary. Primary human adult dermal fibroblasts (HDFa) from three different female donors were obtained from Gibco…
Aiming At MGI High-Throughput Sequencing Platform Fast DNA Library Prep Kit
Product Description 【Product Name】 Fast DNA Library Prep Kit for MGI 【Cat. No./Spec.】 KM001-A/24 rxns; KM001-B/96 rxns; sample sack/ 6 rxns 【Product Description】 Aiming at MGI high-throughput sequencing platform, this kit provides a convenient and universal DNA library construction scheme in one tube. It combines end repair and A-Tailing into…
Optimized minimal genome-wide human sgRNA library
sgRNA collection and enzyme cut site annotation All sgRNAs corresponding to potential NGG-containing target sites on the human (GRCh38/hg38) genome were calculated by a customized Perl script. The enzyme cut site was set at the 17 position from 5’ to 3’ of each sgRNA sequence. These sgRNAs have more than…
Nanopore long-read next-generation sequencing for detection of mitochondrial DNA large-scale deletions
. 2023 Jun 29;14:1089956. doi: 10.3389/fgene.2023.1089956. eCollection 2023. Affiliations Expand Affiliations 1 Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy. 2 Department of Pathophysiology and Transplantation (DEPT), University of Milan, Milan, Italy. Item in Clipboard Chiara Frascarelli et al. Front Genet. 2023. Show details Display…
Isolation and genome characterization of Lloviu virus from Italian Schreibers’s bats
Ethics and biosafety statement Bats were captured during the day with hand-nets in full compliance with best practice and national and European regulations, in derogation to the Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora (authorization 38,025 of 13/08/2020…
Overexpression of human alpha-Synuclein leads to dysregulated microbiome/metabolites with ageing in a rat model of Parkinson disease | Molecular Neurodegeneration
Aim, design and setting of the study We aim to address our research question – how does microbial diversity affect PD pathology with ageing at the molecular level and can early intervention in the microbiome modulate the disease course? Herein, we have studied longitudinally the gut microbiome dynamics with ageing and report that with ageing,…
Isolation and Transcriptome Analysis of Plant Cell Types
The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis. Transcriptome analysis on single cell or cell types can detect the subtle…
Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons
Clinical RNA SARS-CoV-2 isolate For surveillance studies, a set of residual nasopharyngeal swab specimens positive for SARS-CoV-2 qRT-PCR from the Republican Diagnostic Centre (RDC) (umc.org.kz/en/) and private laboratory KDL “Olymp” (www.kdlolymp.kz/) were collected across the Republic of Kazakhstan between 2020 and 2022. 341 sequences that passed quality control are deposited…
A pipeline for sample tagging of whole genome bisulfite sequencing data using genotypes of whole genome sequencing | BMC Genomics
Sample collection and WGS DNA samples were obtained from the CNSR-III [17], a nationwide prospective registry for patients presented to hospitals with acute ischaemic cerebrovascular events between August 2015 and March 2018 in China. Written informed consent was obtained from all patients or legally authorized representatives before entering the study….
Single-cell quantification of ribosome occupancy in early mouse development
PDMS chip fabrication Moulds were 3D-printed by Proto Labs with WaterShed XC 11122 at high resolution (Supplementary Fig. 1). Reusable moulds were assembled by taping 3D-printed molds to glass slides (5″ × 4″; Ted Pella). Sylgard 184 PDMS monomer and curing agent (4019862, Ellsworth Adhesives) were mixed at a 10:1…
Using nanopore sequencing to identify fungi from clinical samples with high phylogenetic resolution
Fungal genomic DNA DNA extracted from Aspergillus niger (kindly provided by Dr. Takamitsu Imoto, Medical Research Institute, Kitano Hospital, Osaka, Japan) and a mock community DNA standard (Mycobiome Genomic DNA Mix, MSA-1010; ATCC, Manassas, VA, USA) were used to evaluate the validity of the sequencing methodology. The mock community standard…
A pangenome reference of 36 Chinese populations
Populations and samples For Phase I of the CPC project, we selected 68 samples from 731 individuals with genomes deep-sequenced using next-generation sequencing. Following a previous study5, we applied a procedure to quantitatively evaluate the genetic diversity coverage based on principal component analysis results. We selected individuals using a statistic…
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…
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…
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…
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…
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…
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…
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…
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…
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…
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….
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)…
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…
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…
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…
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…
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…
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…
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…
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….
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…
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…
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,…
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…
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…
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…
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…
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…
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…
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…
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…
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…
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…
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…
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…
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)…
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…
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…
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…
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,…
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…
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)…
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…
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…
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…