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deep sequencing  (LC Sciences)

 
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    Structured Review

    LC Sciences deep sequencing
    gga-miR-130b directly targets the IBDV genome. (A) Diagram of predicted target sites for miR-130b in IBDV genomic RNA. The seed <t>sequence</t> of miR-130b is underlined and was mutated as indicated by the arrow. (B) miR-130b inhibited target gene expression in a dose-dependent manner. DF-1 cells were cotransfected with luciferase reporter vectors containing wild-type (WT) target sites, pRL-TK, and miR-130b at different concentrations. At 48 h posttransfection, cells were lysed and luciferase reporter gene assays were performed to measure luciferase activities. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miR-130b mimics/luciferase activity of cells cotransfected with the reporter plasmid and miRNA controls. (C) A point mutation in the target gene abolished miR-130b-induced suppression of the target gene. DF-1 cells were cotransfected with miR-130b and the WT or mutant luciferase reporter vector. At 48 h posttransfection, a luciferase reporter gene assay was performed to measure luciferase activity. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
    Deep Sequencing, supplied by LC Sciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "MicroRNA gga-miR-130b Suppresses Infectious Bursal Disease Virus Replication via Targeting of the Viral Genome and Cellular Suppressors of Cytokine Signaling 5"

    Article Title: MicroRNA gga-miR-130b Suppresses Infectious Bursal Disease Virus Replication via Targeting of the Viral Genome and Cellular Suppressors of Cytokine Signaling 5

    Journal: Journal of Virology

    doi: 10.1128/JVI.01646-17

    gga-miR-130b directly targets the IBDV genome. (A) Diagram of predicted target sites for miR-130b in IBDV genomic RNA. The seed sequence of miR-130b is underlined and was mutated as indicated by the arrow. (B) miR-130b inhibited target gene expression in a dose-dependent manner. DF-1 cells were cotransfected with luciferase reporter vectors containing wild-type (WT) target sites, pRL-TK, and miR-130b at different concentrations. At 48 h posttransfection, cells were lysed and luciferase reporter gene assays were performed to measure luciferase activities. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miR-130b mimics/luciferase activity of cells cotransfected with the reporter plasmid and miRNA controls. (C) A point mutation in the target gene abolished miR-130b-induced suppression of the target gene. DF-1 cells were cotransfected with miR-130b and the WT or mutant luciferase reporter vector. At 48 h posttransfection, a luciferase reporter gene assay was performed to measure luciferase activity. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
    Figure Legend Snippet: gga-miR-130b directly targets the IBDV genome. (A) Diagram of predicted target sites for miR-130b in IBDV genomic RNA. The seed sequence of miR-130b is underlined and was mutated as indicated by the arrow. (B) miR-130b inhibited target gene expression in a dose-dependent manner. DF-1 cells were cotransfected with luciferase reporter vectors containing wild-type (WT) target sites, pRL-TK, and miR-130b at different concentrations. At 48 h posttransfection, cells were lysed and luciferase reporter gene assays were performed to measure luciferase activities. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miR-130b mimics/luciferase activity of cells cotransfected with the reporter plasmid and miRNA controls. (C) A point mutation in the target gene abolished miR-130b-induced suppression of the target gene. DF-1 cells were cotransfected with miR-130b and the WT or mutant luciferase reporter vector. At 48 h posttransfection, a luciferase reporter gene assay was performed to measure luciferase activity. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

    Techniques Used: Sequencing, Targeted Gene Expression, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis, Reporter Gene Assay

    The SOCS5 gene is a cellular target of gga-miR-130b. (A) Diagram of predicted target sites for miR-130b in the SOCS5 gene. The seed sequence of miR-130b is underlined and was mutated as indicated by the arrow. (B) Transfection of gga-miR-130b reduced expression of SOCS5. DF-1 cells were cotransfected with miRNAs and luciferase reporter vectors. At 48 h posttransfection, cells were lysed, and a luciferase reporter gene assay was performed to measure SOCS5 expression. The relative level of luciferase activity was calculated as follows: luciferase activity of reporter plasmid-transfected cells or cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. (C) Mutation of the target site abolished the inhibition of SOCS5 by miR-130b. DF-1 cells were cotransfected with miRNA controls or miR-130b mimics or inhibitors and luciferase reporter vectors. At 48 h posttransfection, the assay was performed to measure the luciferase activity. The relative level of luciferase activity was calculated as described above. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001.
    Figure Legend Snippet: The SOCS5 gene is a cellular target of gga-miR-130b. (A) Diagram of predicted target sites for miR-130b in the SOCS5 gene. The seed sequence of miR-130b is underlined and was mutated as indicated by the arrow. (B) Transfection of gga-miR-130b reduced expression of SOCS5. DF-1 cells were cotransfected with miRNAs and luciferase reporter vectors. At 48 h posttransfection, cells were lysed, and a luciferase reporter gene assay was performed to measure SOCS5 expression. The relative level of luciferase activity was calculated as follows: luciferase activity of reporter plasmid-transfected cells or cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. (C) Mutation of the target site abolished the inhibition of SOCS5 by miR-130b. DF-1 cells were cotransfected with miRNA controls or miR-130b mimics or inhibitors and luciferase reporter vectors. At 48 h posttransfection, the assay was performed to measure the luciferase activity. The relative level of luciferase activity was calculated as described above. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001.

    Techniques Used: Sequencing, Transfection, Expressing, Luciferase, Reporter Gene Assay, Activity Assay, Plasmid Preparation, Mutagenesis, Inhibition

    Related Articles

    Sequencing:

    Article Title: gga-miR-155 Enhances Type I Interferon Expression and Suppresses Infectious Burse Disease Virus Replication via Targeting SOCS1 and TANK
    Article Snippet: All miRNA mimics and inhibitors (chemically-modified and single-stranded RNA oligonucleotide that is reverse complement sequence of mature gga-miR-155) were synthesized by GenePharma Company (Shanghai, China). .. Deep sequencing was performed by LC Sciences (Hangzhou, China) on DF-1 cells infected with mock or IBDV at an MOI of 1 for 24 h (accession number of deep sequencing in GEO database: GSE90095 ). miRNA targets in host cells were predicted by TargetScan (version 3.1; Whitehead Institude for Biomedical Research [ http://www.targetscan.org/mamm_31/ ]) and miRanda [August 2010 Release; Memorial Sloan Kettering Cancer Center ( http://www.microrna.org/microrna/home.do )]. .. DF-1 cells were seeded in 24-well or 12-well plates for 24 h, and then miRNA mimics or inhibitors (200 nM) were transfected using Lipofectamine 2000 transfection reagents (invitrogen).

    Article Title: Extracellular vesicles derived from endothelial cells in hypoxia contribute to pulmonary artery smooth muscle cell proliferation in‐vitro and pulmonary hypertension in mice
    Article Snippet: .. Total RNA was isolated using a miRNeasy Mini Kit or miRNeasy Micro Kit (Qiagen) and treated with RNase‐Free DNase I (Qiagen), quantified with Nanodrop 2000 spectrophotometer (Thermo Scientific) and then used for miRNA deep sequencing (LC Sciences) or quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis. .. Six batches of EV from mPVEC exposed to room air or hypoxia (1% O 2 , 24 h) (H‐EV vs. N‐EV) were collected for miRNA deep sequencing (LC Sciences).

    Article Title: Gga-miR-29a-3p suppresses avian reovirus-induced apoptosis and viral replication via targeting Caspase-3.
    Article Snippet: Avian reovirus (ARV) is an important pathogen causing multiple types of clinical diseases in chickens, including viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe.. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post transcriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection.. However, the role of miRNAs in host response to ARV infection is still not clear.

    Article Title: Extracellular vesicles derived from endothelial cells in hypoxia contribute to pulmonary artery smooth muscle cell proliferation in‐vitro and pulmonary hypertension in mice
    Article Snippet: Total RNA was isolated using a miRNeasy Mini Kit or miRNeasy Micro Kit (Qiagen) and treated with RNase‐Free DNase I (Qiagen), quantified with Nanodrop 2000 spectrophotometer (Thermo Scientific) and then used for miRNA deep sequencing (LC Sciences) or quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis. .. Six batches of EV from mPVEC exposed to room air or hypoxia (1% O 2 , 24 h) (H‐EV vs. N‐EV) were collected for miRNA deep sequencing (LC Sciences). .. Briefly, a small RNA library was generated from RNA isolated from N‐EV and H‐EV using the Illumina TruseqTM Small RNA Preparation kit (Illumina).

    Article Title: Identification of benzamides derivatives of norfloxacin as promising microRNA-21 inhibitors via repressing its transcription.
    Article Snippet: MicroRNA-21 is a carcinogenic microRNA, whose overexpression arises in a variety of tumor tissues.. Hence, microRNA-21 a prospective target for cancer treatment, and regulation of microRNA-21 by small molecule inhibitors is deemed as a promising approach for tumor therapy.. In this work, to discover potent microRNA-21 inhibitor, series of 4-(N-norfloxacin-acyl)aminobenzamides were designed and synthesized, and their inhibitory effects were appraised by utilizing dual luciferase reporter assays.

    Infection:

    Article Title: gga-miR-155 Enhances Type I Interferon Expression and Suppresses Infectious Burse Disease Virus Replication via Targeting SOCS1 and TANK
    Article Snippet: All miRNA mimics and inhibitors (chemically-modified and single-stranded RNA oligonucleotide that is reverse complement sequence of mature gga-miR-155) were synthesized by GenePharma Company (Shanghai, China). .. Deep sequencing was performed by LC Sciences (Hangzhou, China) on DF-1 cells infected with mock or IBDV at an MOI of 1 for 24 h (accession number of deep sequencing in GEO database: GSE90095 ). miRNA targets in host cells were predicted by TargetScan (version 3.1; Whitehead Institude for Biomedical Research [ http://www.targetscan.org/mamm_31/ ]) and miRanda [August 2010 Release; Memorial Sloan Kettering Cancer Center ( http://www.microrna.org/microrna/home.do )]. .. DF-1 cells were seeded in 24-well or 12-well plates for 24 h, and then miRNA mimics or inhibitors (200 nM) were transfected using Lipofectamine 2000 transfection reagents (invitrogen).

    Article Title: Gga-miR-29a-3p suppresses avian reovirus-induced apoptosis and viral replication via targeting Caspase-3.
    Article Snippet: Avian reovirus (ARV) is an important pathogen causing multiple types of clinical diseases in chickens, including viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome, leading to considerable economic losses to the poultry industry across the globe.. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post transcriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection.. However, the role of miRNAs in host response to ARV infection is still not clear.

    other:


    Article Title: Identification and Characterization of microRNAs during Retinoic Acid-Induced Regeneration of a Molluscan Central Nervous System
    Article Snippet: Total RNA was extracted using TRI Reagent (Sigma) and Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA), and outsourced to LC Sciences (Houston, TX, USA) for deep sequencing.

    Isolation:

    Article Title: Extracellular vesicles derived from endothelial cells in hypoxia contribute to pulmonary artery smooth muscle cell proliferation in‐vitro and pulmonary hypertension in mice
    Article Snippet: .. Total RNA was isolated using a miRNeasy Mini Kit or miRNeasy Micro Kit (Qiagen) and treated with RNase‐Free DNase I (Qiagen), quantified with Nanodrop 2000 spectrophotometer (Thermo Scientific) and then used for miRNA deep sequencing (LC Sciences) or quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis. .. Six batches of EV from mPVEC exposed to room air or hypoxia (1% O 2 , 24 h) (H‐EV vs. N‐EV) were collected for miRNA deep sequencing (LC Sciences).

    Spectrophotometry:

    Article Title: Extracellular vesicles derived from endothelial cells in hypoxia contribute to pulmonary artery smooth muscle cell proliferation in‐vitro and pulmonary hypertension in mice
    Article Snippet: .. Total RNA was isolated using a miRNeasy Mini Kit or miRNeasy Micro Kit (Qiagen) and treated with RNase‐Free DNase I (Qiagen), quantified with Nanodrop 2000 spectrophotometer (Thermo Scientific) and then used for miRNA deep sequencing (LC Sciences) or quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis. .. Six batches of EV from mPVEC exposed to room air or hypoxia (1% O 2 , 24 h) (H‐EV vs. N‐EV) were collected for miRNA deep sequencing (LC Sciences).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Extracellular vesicles derived from endothelial cells in hypoxia contribute to pulmonary artery smooth muscle cell proliferation in‐vitro and pulmonary hypertension in mice
    Article Snippet: .. Total RNA was isolated using a miRNeasy Mini Kit or miRNeasy Micro Kit (Qiagen) and treated with RNase‐Free DNase I (Qiagen), quantified with Nanodrop 2000 spectrophotometer (Thermo Scientific) and then used for miRNA deep sequencing (LC Sciences) or quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis. .. Six batches of EV from mPVEC exposed to room air or hypoxia (1% O 2 , 24 h) (H‐EV vs. N‐EV) were collected for miRNA deep sequencing (LC Sciences).



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    Image Search Results


    RADDIM creates random duplications or deletions at one random position within a circular DNA molecule. ( A ) Illustration of the RADDIM workflow when starting from a plasmid template. Illustration created with BioRender.com . ( B ) An ExoChase-treated pUC19 plasmid (Fig. ) containing a CcdB toxin gene was nicked on the forward DNA-strand by the site-specific nickase Nt.BbvCI. The resulting double-nicked plasmids were incubated with Bst DNAP, with or without either the 5′–3′ single-strand-specific RecJ exonuclease, or the 3′–5′ single-strand-specific Thermolabile Exonuclease I, to determine if single-strand exonucleases could remove unwanted DNA-amplifications during a convergent nick-translation reaction. ( C ) Characterization of the DNA-ends that were created by a convergent nick-translation reaction using Bst DNAP and either RecJ or Thermolabile Exo I (Fig. ). ConNickTra linearized plasmids were purified and T7 DNAP was used to repair all DNA-ends. Next, T4 DNA ligase was used to re-circularize the linear plasmids, which were transformed into CcdB-sensitive E. coli cells. This selected for plasmids with a mutation in the ccdB toxin gene. The ccdB gene of 40 successfully sequenced plasmids from the RecJ library ( n = 40), and 39 successfully sequenced plasmids from the Thermolabile Exo I library ( n = 39) were sequenced by single-colony Sanger sequencing. ( D ) A synthetic 196 bp dsDNA fragment was incubated together with Bst DNAP alone, and/or Thermolabile Exo I, for 3 h with or without 1 mM MnCl 2 , to evaluate if manganese could reprogram the Bst DNAP and/or ExoI to degrade the ends of linear DNA molecules. ( E ) A pUC19 plasmid containing a CcdB toxin gene was randomly linearized by a ConNickTra reaction using Bst DNAP and ExoI, followed by an addition of 1 mM MnCl 2 for 10–30 min. After a T7 DNAP-mediated DNA end-repair, and a T4 DNA ligase-mediated re-circularization, plasmids were transformed into CcdB-sensitive E. coli cells. ( F ) The ccdB gene of 25 successfully sequenced plasmids from the 10 min library ( n = 25), 21 plasmids from the 20 min library ( n = 21), and 22 successfully sequenced plasmids from the 30 min library ( n = 22) were analyzed by single-colony Sanger sequencing.

    Journal: Nucleic Acids Research

    Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs

    doi: 10.1093/nar/gkag236

    Figure Lengend Snippet: RADDIM creates random duplications or deletions at one random position within a circular DNA molecule. ( A ) Illustration of the RADDIM workflow when starting from a plasmid template. Illustration created with BioRender.com . ( B ) An ExoChase-treated pUC19 plasmid (Fig. ) containing a CcdB toxin gene was nicked on the forward DNA-strand by the site-specific nickase Nt.BbvCI. The resulting double-nicked plasmids were incubated with Bst DNAP, with or without either the 5′–3′ single-strand-specific RecJ exonuclease, or the 3′–5′ single-strand-specific Thermolabile Exonuclease I, to determine if single-strand exonucleases could remove unwanted DNA-amplifications during a convergent nick-translation reaction. ( C ) Characterization of the DNA-ends that were created by a convergent nick-translation reaction using Bst DNAP and either RecJ or Thermolabile Exo I (Fig. ). ConNickTra linearized plasmids were purified and T7 DNAP was used to repair all DNA-ends. Next, T4 DNA ligase was used to re-circularize the linear plasmids, which were transformed into CcdB-sensitive E. coli cells. This selected for plasmids with a mutation in the ccdB toxin gene. The ccdB gene of 40 successfully sequenced plasmids from the RecJ library ( n = 40), and 39 successfully sequenced plasmids from the Thermolabile Exo I library ( n = 39) were sequenced by single-colony Sanger sequencing. ( D ) A synthetic 196 bp dsDNA fragment was incubated together with Bst DNAP alone, and/or Thermolabile Exo I, for 3 h with or without 1 mM MnCl 2 , to evaluate if manganese could reprogram the Bst DNAP and/or ExoI to degrade the ends of linear DNA molecules. ( E ) A pUC19 plasmid containing a CcdB toxin gene was randomly linearized by a ConNickTra reaction using Bst DNAP and ExoI, followed by an addition of 1 mM MnCl 2 for 10–30 min. After a T7 DNAP-mediated DNA end-repair, and a T4 DNA ligase-mediated re-circularization, plasmids were transformed into CcdB-sensitive E. coli cells. ( F ) The ccdB gene of 25 successfully sequenced plasmids from the 10 min library ( n = 25), 21 plasmids from the 20 min library ( n = 21), and 22 successfully sequenced plasmids from the 30 min library ( n = 22) were analyzed by single-colony Sanger sequencing.

    Article Snippet: The RADDIM PacBio Revio deep sequencing data analysis and the Python scripts used to calculate the theoretical mutational landscapes possible with NSM are available from the Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) GitHub for this project ( https://github.com/biosustain/raddim ) and on Zenodo ( https://doi.org/10.5281/zenodo.18863538 ).

    Techniques: Plasmid Preparation, Incubation, Nick Translation, Purification, Transformation Assay, Mutagenesis, Sequencing

    RADDIM allows for in-frame and multi-residue InDels enabling functional protein structure modifications. ( A ) Illustration of an alternative RADDIM workflow to insert random DNA sequences into a RADDIM library by ligating a random DNA sequence oligo library to ConNickTra linearized plasmids, followed by a T7 DNAP-mediated DNA-end-repair/fill-in. Illustration created with BioRender.com . ( B ) Representative β-lactamase compensatory mutations able to restore phenotypic ampicillin resistance of the enzymatically inactivated (A40P and R41W) TEM-1 protein, superimposed onto the wild-type TEM-1 protein structure (PDB: 1ZG4). Red spheres = original inactivating mutations (A40P and R41W), Green spheres = compensatory AA substitutions. Purple marking = site of multi-residue compensatory deletion. Brown marking = site of multi-residue compensatory insertions.

    Journal: Nucleic Acids Research

    Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs

    doi: 10.1093/nar/gkag236

    Figure Lengend Snippet: RADDIM allows for in-frame and multi-residue InDels enabling functional protein structure modifications. ( A ) Illustration of an alternative RADDIM workflow to insert random DNA sequences into a RADDIM library by ligating a random DNA sequence oligo library to ConNickTra linearized plasmids, followed by a T7 DNAP-mediated DNA-end-repair/fill-in. Illustration created with BioRender.com . ( B ) Representative β-lactamase compensatory mutations able to restore phenotypic ampicillin resistance of the enzymatically inactivated (A40P and R41W) TEM-1 protein, superimposed onto the wild-type TEM-1 protein structure (PDB: 1ZG4). Red spheres = original inactivating mutations (A40P and R41W), Green spheres = compensatory AA substitutions. Purple marking = site of multi-residue compensatory deletion. Brown marking = site of multi-residue compensatory insertions.

    Article Snippet: The RADDIM PacBio Revio deep sequencing data analysis and the Python scripts used to calculate the theoretical mutational landscapes possible with NSM are available from the Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) GitHub for this project ( https://github.com/biosustain/raddim ) and on Zenodo ( https://doi.org/10.5281/zenodo.18863538 ).

    Techniques: Residue, Functional Assay, Sequencing

    Deep sequencing confirms the diversity of RADDIM-generated InDel libraries. ( A ) Size distribution of insertions and deletions across a RADDIM plasmid library and the location of all variants (insertions and deletions) that are 1 nt and >1 nt in length. InDels are shown by their start position in the 5′–3′ direction in the plasmid sequence. Positive values represent insertions and negative values represent deletions. CAT = chloramphenicol acetyltransferase, tCYC1 = transcriptional terminator of iso-1-cytochrome c from S. cerevisiae , ori = pUC19 origin-of-replication, BLA* = inactivated (A40P and R41W) β-lactamase (TEM-1), CcdB = bacterial DNA gyrase toxin, CcdA* = inactivated cognate immunity protein of CcdB. ( B ) Illustration of the plasmid linearization mechanisms attained by combining the ExoChase and ConNickTra methods, enabling random and singular double-stranded DNA-breaks to be enriched within only one half of a plasmid molecule, down-stream of the site-specific DNA-nick. Illustration created with BioRender.com . ( C ) Quantification of all identified deletions ranging from 1 to 30 nt in length. ( D ) Quantification of all identified insertions ranging from 1 to 30 nt in length. ( E ) The number of identified mismatches for all insertions ranging from 2 to 30 nt in length.

    Journal: Nucleic Acids Research

    Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs

    doi: 10.1093/nar/gkag236

    Figure Lengend Snippet: Deep sequencing confirms the diversity of RADDIM-generated InDel libraries. ( A ) Size distribution of insertions and deletions across a RADDIM plasmid library and the location of all variants (insertions and deletions) that are 1 nt and >1 nt in length. InDels are shown by their start position in the 5′–3′ direction in the plasmid sequence. Positive values represent insertions and negative values represent deletions. CAT = chloramphenicol acetyltransferase, tCYC1 = transcriptional terminator of iso-1-cytochrome c from S. cerevisiae , ori = pUC19 origin-of-replication, BLA* = inactivated (A40P and R41W) β-lactamase (TEM-1), CcdB = bacterial DNA gyrase toxin, CcdA* = inactivated cognate immunity protein of CcdB. ( B ) Illustration of the plasmid linearization mechanisms attained by combining the ExoChase and ConNickTra methods, enabling random and singular double-stranded DNA-breaks to be enriched within only one half of a plasmid molecule, down-stream of the site-specific DNA-nick. Illustration created with BioRender.com . ( C ) Quantification of all identified deletions ranging from 1 to 30 nt in length. ( D ) Quantification of all identified insertions ranging from 1 to 30 nt in length. ( E ) The number of identified mismatches for all insertions ranging from 2 to 30 nt in length.

    Article Snippet: The RADDIM PacBio Revio deep sequencing data analysis and the Python scripts used to calculate the theoretical mutational landscapes possible with NSM are available from the Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) GitHub for this project ( https://github.com/biosustain/raddim ) and on Zenodo ( https://doi.org/10.5281/zenodo.18863538 ).

    Techniques: Sequencing, Generated, Plasmid Preparation

    RADDIM enables a random duplication or deletion of entire regulatory DNA motifs. ( A ) Illustration of the last steps in the RADDIM workflow when starting from a linear PCR-product (Fig. ). Illustration created with BioRender.com . Relative mNeonGreen fluorescent protein expression by S. cerevisiae cells transformed with RADDIM-mutated ( B ) pACT1 ( n = 90) or ( C ) pTEF1 promoter variants ( n = 86) following a FACS of top 1% of fluorescent cells. ( D ) Relative mNeonGreen fluorescent protein expression by reconstituted pACT1 and pTEF1 promoter variants ( n = 3). Statistical significance was calculated by two-way ANOVA with ns: P > 0.05, *: P ≤ 0.05, **: P ≤ 0.005, ***: P ≤ 0.0005, and ****: P ≤ 0.0001. ( E) Relative mNeonGreen fluorescent protein expression by wild-type pACT1 and pTEF1 promoters ( n = 3). Statistical significance was calculated by unpaired t -test with ns: P > 0.05 and *: P ≤ 0.0001.

    Journal: Nucleic Acids Research

    Article Title: High-throughput methods enabling random duplications, deletions, or nucleotide-constrained mutagenesis of entire DNA motifs

    doi: 10.1093/nar/gkag236

    Figure Lengend Snippet: RADDIM enables a random duplication or deletion of entire regulatory DNA motifs. ( A ) Illustration of the last steps in the RADDIM workflow when starting from a linear PCR-product (Fig. ). Illustration created with BioRender.com . Relative mNeonGreen fluorescent protein expression by S. cerevisiae cells transformed with RADDIM-mutated ( B ) pACT1 ( n = 90) or ( C ) pTEF1 promoter variants ( n = 86) following a FACS of top 1% of fluorescent cells. ( D ) Relative mNeonGreen fluorescent protein expression by reconstituted pACT1 and pTEF1 promoter variants ( n = 3). Statistical significance was calculated by two-way ANOVA with ns: P > 0.05, *: P ≤ 0.05, **: P ≤ 0.005, ***: P ≤ 0.0005, and ****: P ≤ 0.0001. ( E) Relative mNeonGreen fluorescent protein expression by wild-type pACT1 and pTEF1 promoters ( n = 3). Statistical significance was calculated by unpaired t -test with ns: P > 0.05 and *: P ≤ 0.0001.

    Article Snippet: The RADDIM PacBio Revio deep sequencing data analysis and the Python scripts used to calculate the theoretical mutational landscapes possible with NSM are available from the Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) GitHub for this project ( https://github.com/biosustain/raddim ) and on Zenodo ( https://doi.org/10.5281/zenodo.18863538 ).

    Techniques: Expressing, Transformation Assay

    Profiling of ovarian senescence-associated miRNA expression by transcriptome sequencing across multiple laying ages (350–700 d). (A) PCA revealed a distinct separation among the three groups, n = 3. (B) Heatmap of top 30 DEMs across the period, n = 3. (C-E) Volcano plot depicted DEMs in pairwise comparisons among the 350 d, 500 d, and 700 d groups, n = 3.

    Journal: Poultry Science

    Article Title: miRNA profiling reveals that gga-let-7i/COL1A2 axis induces cell cycle arrest and triggers cellular senescence to accelerate ovarian aging in laying hens by suppressing the PI3K/AKT/MDM2 pathway

    doi: 10.1016/j.psj.2026.106542

    Figure Lengend Snippet: Profiling of ovarian senescence-associated miRNA expression by transcriptome sequencing across multiple laying ages (350–700 d). (A) PCA revealed a distinct separation among the three groups, n = 3. (B) Heatmap of top 30 DEMs across the period, n = 3. (C-E) Volcano plot depicted DEMs in pairwise comparisons among the 350 d, 500 d, and 700 d groups, n = 3.

    Article Snippet: Gene Ontology ( GO ) enrichment and Kyoto Encyclopedia of Genes and Genomes ( KEGG ) pathway analysis were conducted on the target gene candidates of DEMs through ClusterProfile (for GO analysis) and KOBAS (for KEGG analysis) software. miRNA library construction and deep sequencing were performed by Novogene (Beijing, China).

    Techniques: Expressing, Sequencing