Viral DNA from a chronic hepatitis B patient was isolated from 100 μl of serum using the QIAamp UltraSens Virus kit (Qiagen, Venlo, Limburg, Netherlands). Full-length HBV amplicons were obtained by PCR amplification of 5 ng of viral DNA using previously published primers [53 (link)], gel-purified using a MinElute Gel Extraction kit (Qiagen), cloned into a pCR2.1-TOPO vector (Life Technologies, Carlsbad, CA, USA), and transformed into Escherichia coli ABLE K competent cells (Agilent, Santa Clara, CA, USA) following the manufacturers’ protocols. Purified clones were verified for the presence of an approximately 3.2 kb insert by PCR, and full-length sequencing of the insert was performed using a primer walking approach (Table S1 in Additional file 1 ). The sequencing reactions were performed using a BIGDYE Terminator v3.1 kit (Life Technologies) and loaded on a 3730xl instrument (Life Technologies) for analysis. For BAsE-Seq library preparation, each HBV clone (Clone-1 and Clone-2) was linearized by restriction digest with NotI (NEB, Ipswich, MA, USA), gel-purified using a MinElute Gel Extraction kit (Qiagen), quantified using a Qubit dsDNA BR assay kit (Life Technologies), and diluted to 106 copies/μl.
Minelute gel extraction kit
Manufactured by Qiagen
Sourced in Germany, United States, France, United Kingdom, Netherlands, China
The MinElute Gel Extraction Kit is a laboratory instrument designed to extract and purify DNA fragments from agarose gels. It utilizes a silica membrane technology to efficiently capture and concentrate the target DNA.
Automatically generated - may contain errors
Lab products found in correlation
Minelute pcr purification kit, by Qiagen (13 mentions)
Ampure xp bead, by Beckman Coulter (11 mentions)
Miseq platform, by Illumina (10 mentions)
Qiaquick pcr purification kit, by Qiagen (9 mentions)
Hiseq 2000, by Illumina (8 mentions)
Qiaprep spin miniprep kit, by Qiagen (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Miseq reagent kit v3, by Illumina (7 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (6 mentions)
Epitect bisulfite kit, by Qiagen (6 mentions)
Nextseq 500, by Illumina (5 mentions)
Miseq sequencer, by Illumina (5 mentions)
Hiseq 2500, by Illumina (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Pgem t easy vector, by Promega (5 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (5 mentions)
Agencourt ampure xp bead, by Beckman Coulter (4 mentions)
Onestep rt pcr kit, by Qiagen (4 mentions)
Truseq rna sample preparation kit v2 set a, by Illumina (4 mentions)
Qubit dna br assay kit, by Thermo Fisher Scientific (4 mentions)
290 protocols using minelute gel extraction kit
1
HBV Amplification and Sequencing Protocol
2
Virus dsRNA Extraction from Stool
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Virus dsRNA was extracted from the stool samples using the TRIzol™ reagent, as previously described (Potgieter et al., 2009 (link)), albeit with minor modifications. Briefly, approximately 500 mg of fresh stool was added to 1 mL of TRIzol™ reagent (Invitrogen, Carlsbad, CA) and incubated at room temperature for 5 min; and then centrifuged at 16,000 RPM for 15 min at 4°C (same centrifugation conditions for the next steps). Thereafter, 300 μL of chloroform (Sigma-Aldrich®, St. Louis, MO, United States) was added to the solution, followed by centrifugation. The supernatant was transferred to a clean 2-ml tube, 650 μL of isopropanol (Sigma-Aldrich®, St. Louis, MO, United States) was added, and the tube was centrifuged. The supernatant was poured off, and the tubes dried for 5 min, then the pellet was eluted with 95 μL of elution buffer (EB) (MinElute Gel extraction kit-Qiagen, Hilden, Germany). Approximately 30 μL of 8 M lithium chloride (Sigma, St. Louis, MO, USA) was added to the RNA, incubated at 4°C overnight for precipitation, and then centrifuged at 16000 x g at 4°C for 30 min. The mixture was purified using the MinElute Gel extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purified product was verified by electrophoresis in a 1.0% agarose gel under ultraviolet light (Bio-Rad Laboratories, Hercules, CA, United States).
3
ACE2 Gene Expression Analysis by PCR
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ACE2 PCR was performed with cDNA obtained as described earlier. Fifty nanograms of cDNA was used in the following reaction: initial denaturation – 3 minutes, 98oC and 35 cycles of denaturation – 30 seconds at 98oC, annealing – 30 seconds at 58oC, extension – 72oC for 2 minutes, ending with final extension of 72oC for 10 minutes. Amplified fragments were run on a 1.5% agarose in 1x TAE gel with 100-kb DNA ladder to assess product size. Bands were cut out, and PCR products cleaned with MinElute Gel Extraction Kit (Quiagen) and Sanger sequenced by Quintara Biosciences. Primers used were as follows: dACE2 forward: 5’-TGTGAGAGCCTTAGGTTGGATTCC-3’, dACE2 reverse: 5’-TCTCTCCTTGGCCATGTTGT-3’ (Onabajo et al., 2020 (link)).
4
Quantifying ACE2 Expression via PCR
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ACE2 PCR was performed with cDNA obtained as described above. 50ng of cDNA was used in the following reaction: initial denaturation – 3 minutes, 98°C and 35 cycles of denaturation – 30 seconds at 98°C, annealing – 30 seconds at 58°C, extension – 72°C for 2 minutes, ending with final extension of 72°C for 10 minutes. Amplified fragments were run on a 1.5% agarose in 1xTAE gel with 100 kb DNA ladder to assess product size. Bands were cut out and PCR products cleaned with MinElute Gel Extraction Kit (Quiagen) and Sanger sequenced by Quintara Biosciences. Primers used: dACE2 forward: 5’-TGTGAGAGCCTTAGGTTGGATTCC-3’, dACE2 reverse: 5’-TCTCTCCTTGGCCATGTTGT-3’. (Onabajo et al., 2020 )
5
Illumina Sequencing of Five Individuals
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Illumina sequencing of five individuals (three males and two females) was conducted at the Center for Biotechnology (CeBiTec) at Bielefeld University. Libraries were prepared with the Nextera DNA Sample Preparation Kit (Illumina, Little Chesterford, UK) according to the manufacturer's instructions. The DNA was then run on a 1.5% agarose gel and fragments in the size range 600–1000 bp were extracted with the Qiagen MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). Fragment sizes were checked using a High Sensitivity DNA Chip on the Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany). Quantification was performed using the Quant‐iT Picogreen® dsDNA Assay Kit (Life Technologies, Darmstadt, Germany). The libraries were then sequenced on an Illumina MiSeq sequencer using a MiSeq® Reagent Kit v3 (600 cycles; Illumina) to generate 301 bp paired‐end reads. FastQ files were generated automatically by the software MiSeq Reporter(version 2.5.1.3: Illumina Inc, 5200 Illumina Way, 92122 San Diego, CA, USA). Analysis within FastQC (Andrews) indicated that the reads were of high quality.
6
Multiomics Analysis of Cell Types
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Each RNA-seq, ATAC-seq, and MethylC-seq analysis was conducted on two biological replicates except for brain EC ATAC-seq, which was conducted on three biological replicates. Single-cell RNA-seq was conducted with a single sample. Libraries for RNA-seq and ATAC-seq were prepared as previously described, with minor modifications (Buenrostro et al., 2015 (link); Lister et al., 2013 (link); Mo et al., 2015 (link); Mo et al., 2016 (link)). For RNA-seq, total RNA was converted to cDNA and amplified (Ovation Ultralow System V2-32, 0342HV, NuGEN Technologies, San Carlos, CA). Amplified cDNA was fragmented, end-repaired, linker-adapted, and single-end sequenced for 75 cycles on a NextSeq500 (Illumina Inc.). Tagmented DNA was purified using QIAGEN MinElute GelExtraction kit (28604, Qiagen). ATAC-seq libraries were PCR amplified for 11 cycles. Agencourt AMPure XP beads (A63880, Beckman Coulter) were used to purify ATAC-seq libraries, which were then paired-end sequenced for 36 cycles on a NextSeq500. MethylC-seq library preparation discussed below.
7
Bacterial Identification and Characterization
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Salmonella typhimurium (KCCM 12041), Salmonellaenteritidis (KCCM 12021), Escherichiacoli (KCCM 11234), and Staphylococcusaureus (KCCM 12103) were obtained from the Korean Collection of Type Cultures (Daejun, Korea). Tryptic soy (TS) agar, BBL eosin methylene blue (EMB) agar, XLT4 agar base, XLT4 agar supplement, Baird–Parker agar base, and EY tellurite enrichment were purchased from BD Difco (Sparks, MD, USA). The initial ssDNA library and the primers used for amplification were synthesized and purified by polyacrylamide gel electrophoresis (PAGE; Bioneer Co., Ltd, Daejeon, Korea). Phosphate–buffered saline (PBS, pH 7.4) was purchased from Sigma (St. Louis, MO, USA). PCR tubes, reagents, and polymerase were obtained from Takara (Shiga, Japan). LE agarose and TAE buffer were purchased from Lonza (Rockland, ME, USA). The Qiagen MinElute gel extraction kit was obtained from Qiagen (Hilden, Germany). The In-Fusion HD Cloning kit was purchased from Clontech (Mountain View, CA, USA).
8
Generating Heteroduplex DNA Substrates
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pT7Blue-3Rev-TGA and control pT7Blue-3Rev-CGA plasmids are derivatives of pT7Blue-3 AmpR, KamR (Novagen, EMD Millipore, MA, USA), they contain reversed f1 origin and either TGA stop codon or CGA-Arg codon, respectively, inserted after Met19 of kanamycin resistance gene. Note that, pT7Blue-3 and its derivatives do not have any mammalian replication origins and cannot replicate in MEFs. The plasmid vectors were obtained by a polymerase chain reaction-based site-directed mutagenesis. Circular heteroduplex DNA substrate pT7Blue-3Rev-TGA-Hx containing Hx opposite to T within TGA codon (TpCpHx/TpGpA context) was constructed by primer extension, using 5′-phosphorylated pHx-Kan29 d(pTCAGCATCTCHxCATGTTGGAATTTAATCG) oligonucleotide containing single Hx residue as a primer and single-stranded phagemid DNA as a template, as described previously (50 (link),51 (link)). After synthesis of a second strand and ligation, the covalently closed circular heteroduplex plasmid DNA was agarose gel purified using Qiagen MinElute Gel Extraction Kit (Qiagen, France).
9
RNA-seq and ATAC-seq Library Preparation
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Libraries for RNA-seq and ATAC-seq were prepared as previously described (Buenrostro et al., 2015 (link); Sabbagh et al., 2018 (link)). Adult brain EC RNA-seq replicates were single-end sequenced for 75 cycles on a NextSeq500 (Illumina). WT cultured brain EC RNA-seq libraries for replicates one, two, five, and six and beta-catenin stabilized brain EC RNA-seq libraries for all four replicates were paired-end sequenced for 36 cycles on a NextSeq500. WT cultured brain EC RNA-seq libraries for replicates three and four were prepared by Omega Bioservices (Georgia) and paired-end sequenced for 150 cycles. Tagmented DNA was purified using QIAGEN MinElute Gel Extraction kit (28604, Qiagen). ATAC-seq libraries were PCR amplified for 11 cycles. Agencourt AMPure XP beads (A63880, Beckman Coulter) were used to purify ATAC-seq libraries, which were then paired-end sequenced for 36 cycles on a NextSeq500 (Illumina). Sequencing libraries that contained overrepresented adaptor sequences were trimmed using Trim Galore (https://github.com/FelixKrueger/TrimGalore ).
10
Characterizing OrhR-DNA Binding Interactions
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To study the binding of OrhR to DNA probes, EMSAs were performed using a LightShift Chemiluminescent EMSA Kit (Thermo) [60 (link)]. The OrhR-His6 fusion protein was purified to homogeneity using Ni-NTA Spin Columns (Qiagen) and dialyzed against the binding buffer. The DNA probes were PCR amplified using biotinylated primers and gel purified using a Qiagen MinElute Gel Extraction Kit (Qiagen). EMSAs were performed by adding increasing amounts of purified OrhR-His6 fusion protein to the labeled probe (2 fmol) in the binding buffer (10 mM Tris (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 90 mM KCl, 10 mM MgCl2, 10 mM acetyl phosphate, 50 ng/μL poly (dI-dC), 1 μg/mL bovine serum albumin, 5% glycerol) for a 30-min incubation at room temperature. Unlabeled cold probes were added at the ratios indicated. The reaction mixtures were then subjected to electrophoresis on a 6% polyacrylamide gel in 0.5× TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.0) at 100 V for 120 mins. The gel was transferred to a nylon membrane and detected according to the kit instructions, and the image was visualized and captured under a ProteinSimple FluorChem imager.
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