Journal: Cell genomics

Article Title: Direct detection of RNA modifications and structure using single-molecule nanopore sequencing

doi: 10.1016/j.xgen.2022.100097

Figure Lengend Snippet: A) Scheme for direct RNA nanopore sequencing. rRNAs containing native modifications are poly(A) tailed before ligation of adapters and RT. Ionic current blockage events are characteristic of the kmer sequence of RNA transiting through the pore constriction. (B) Read quality heatmap for IVT rRNA (left) and native rRNA (right) from E. coli . Dashed red lines indicated the expected lengths for 16S rRNA (1.5 kb) and 23S rRNA (2.9 kb) (C) Normalized native (red) and IVT (black) current signal alignment for 16S rRNA from E. coli spanning positions 1395–1415 performed using Tombo. Sites of known modifications within this window are highlighted in blue. (D) Positional Kolmogorov-Smirnov (KS) statistical testing of current signals across rRNA from E. coli and S. cerevisiae using both Tombo and Nanopolish. Modification positions described in the literature are indicated as black lines. (E) Median current and dwell KS statistic profiles separated by modification type (base [excluding Ψ ], blue; 2'- O -methyl, red; and Ψ , green) and aligned by modification position from both Tombo and Nanopolish. Colored, shaded regions represent the standard deviation of the KS statistic.

Article Snippet: Direct RNA nanopore sequencing kit , Oxford Nanopore Technologies , SQK-RNA002.

Techniques: Nanopore Sequencing, Ligation, Sequencing, Modification, Standard Deviation

Journal: Cell genomics

Article Title: Direct detection of RNA modifications and structure using single-molecule nanopore sequencing

doi: 10.1016/j.xgen.2022.100097

Figure Lengend Snippet: (A) Scheme for 3' end modification, SHAPE probing, and 3' end tailing. (B) Normalized SHAPE-MaP reactivity (blue) and nanoSHAPE reactivity detected by changes in current (red) and dwell time (gray) for the pri-miR-17~92 RNA. (C) Heatmap of 1,000 nanopore reads of pri-miR-17~92 modified with 150 mM AcIm. Modifications were determined by per-nucleotide Student’s t test using Fisher’s method context of ±1 of the current signal. Per-nucleotide p values were corrected using the Benjamini-Hochberg procedure and binarized. Nucleotides scored as modified and unmodified are shown in black and teal, respectively; unmapped regions are gray. (D) Kernel density estimate of the number of called modifications per pri-miR-17~92 molecule as a function of AcIm concentration. Called modifications correspond to an upper limit. (E) Spearman’s rank-order correlation (rho) between nanoSHAPE and SHAPE-MaP as a function of the number of contributing pri-miR-17~92 molecules across the AcIm concentrations tested. (F) Spearman’s rho as a function of the distance from the 3' end of the pri-miR-17~92 RNA.

Article Snippet: Direct RNA nanopore sequencing kit , Oxford Nanopore Technologies , SQK-RNA002.

Techniques: Modification, Concentration Assay

Journal: Cell genomics

Article Title: Direct detection of RNA modifications and structure using single-molecule nanopore sequencing

doi: 10.1016/j.xgen.2022.100097

Figure Lengend Snippet:

Article Snippet: Direct RNA nanopore sequencing kit , Oxford Nanopore Technologies , SQK-RNA002.

Techniques: Virus, Recombinant, Nanopore Sequencing, Sequencing, Modification, Software

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Schematic diagram of recombinant plasmid pEP-BGH-GAstV ORF2

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Recombinant, Plasmid Preparation

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Recombinant pDEV-GAstV ORF2 construction. pDEV-EF1 (shown in the dotted box ( A and B )) were constructed and detailed in our prior publication . A . Homologous recombination was used to introduce a mini-F BAC vector (pHA2) that allows for large circular DNA to be maintained in E. coli into the intergenic region between UL15B and UL18 in a DEV vaccine strain. B . Two-step Red-mediated recombination ( en passant ) was used to substitute the P CMV promoter controlling GFP expression in pDEV-vac for the pEF1 gene. C - D . The recombinant BAC clone pDEV-kan.GAstV ORF2 was constructed by first inserting P CMV -GAstV ORF2-BGH-pA-Kan expression cassette into the US7 and US8 intergenic spacer in pDEV-EF1( C ) and pDEV-kan.GAstV ORF2 ( D ) were obtained by deleting Kan gene from pDEV-kan.GAstV ORF2 by 2nd homologous recombinant

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Recombinant, Construct, Homologous Recombination, Introduce, Plasmid Preparation, Expressing

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Bam HI digestion and PCR-based identification of recombinant BAC clones. a . Bam HI digestion-based analysis of pDEV-GAstV ORF2 mutants. 1: pDEV-EF1; 2: pDEV-Kan.GAstV ORF2; 3: pDEV-GAstV ORF2 mutants. M: 15,000 bp marker; 1: rDEV-EF1; 2: pDEV-Kan. GAstV ORF2; 3: pDEV-GAstV ORF2. b . PCR-based recombinant mutated BAC clone identification. 1: pDEV-EF1 (641 bp); 2: pDEV-Kan.GAstV ORF2 (4234 bp); 3 pDEV-GAstV ORF2 (3818 bp); M: 250 bp marker (4500, 3000, 2250, 1500, 1000, 750, 500, 250)

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Recombinant, Clone Assay, Marker

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Rescued of recombinant virus. Fluorescence plaque of the rescued rDEV-GAstV ORF2 (100×)

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Recombinant, Virus, Fluorescence

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Plaque size of rDEV-GAstV ORF2 and control virus rDEV-EF1 on CEFs. Image J was used to compute means and standard deviations when measuring the sizes of 100 plaques, setting the mean plaque size for rDEV-EF1 to 100%. Error bars represent the standard deviation. The P value was calculated by the independent sample t -test, and a P value less than 0.05 was regarded as significant

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Control, Virus, Standard Deviation

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Multi-step growth curves of rDEV-GAstV ORF2 amd rDEV-EF1 on CEFs. Comparison of the in vitro growth of viruses reconstructed with parental DEV. The virus titers of infectedcells (A) and supernatants (B) were determined at different times (0, 12, 24, 36, 48, 60, and 72 h) after inoculation of approximately 0.02 MOI of cell-free viruses of rDEV-GAstV ORF2 and rDEV-EF1. The multi-step growth curves were computed from three independent experiments

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Comparison, In Vitro, Virus

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Analyses of GAstV Cap protein expression in virus-infected CEFs and supernatant samples by Western blot analysis. Detection of GAstV ORF2 protein ( A ) and inner protein DEV US3 ( C ) by Western blot analysis. Corresonding picture of SDS-PAGE ( C ). M: Prestained Protein Ladder (10-180 kDa); 1: rDEV-EF1-infected culture supernatants; 2: rDEV-GAstV ORF2-infected culture supernatants; 3: rDEV-EF1-infected CEFs; 4: rDEV-GAstV ORF2-infected CEFs

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Expressing, Virus, Infection, Western Blot, SDS Page

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Analysis of GAstV Cap protein expression and localizaiton in CEFs by IFA. rDEV-GAstV ORF2 or control virus rDEV-EF1-infected CEFs were fixed and taken to IFA performed with anti-GAstV Cap mAb as primary antibody and Cy3-labelled goat anti-mouse IgG as secondary antibody. And then staining with DAPI solution. Red florescence represent Cap protien

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Expressing, Control, Virus, Infection, Staining

Journal: BMC Veterinary Research

Article Title: In vitro expression of the goose astrovirus Cap protein delivered with a duck enteritis virus vector

doi: 10.1186/s12917-025-04654-7

Figure Lengend Snippet: Immunogold labeling electron microscopy analysis of Cap protein in rDEV-GAstV ORF2-infected CEFs. Nucleoprotein (N) immunogold labeling was achieved with an antibody against the Cap protein. GAstV Cap proteins are denoted by arrows pointing to colloidal gold spots. Scale bar: 200 nm; VLPs are marked with asterisks; Scale bar: 500 nm

Article Snippet: GAstV ORF2 containing separate Not I and Kpn I sites introduced into the 5’and 3’ termini was optimized according to the duck codon preferences, after which it was synthesized by GeneScript (Nanjing, China) with reference to the GAstV gene sequence (GenBank MH052598).

Techniques: Labeling, Electron Microscopy, Infection