1 kb downstream regions  (New England Biolabs)


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    New England Biolabs 1 kb downstream regions
    MspI
    MspI 25 000 units
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    Average 86 stars, based on 3506 article reviews
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    1 kb downstream regions - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius"

    Article Title: Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius

    Journal: Journal of Industrial Microbiology & Biotechnology

    doi: 10.1007/s10295-014-1415-6

    Gel electrophoresis of SQ-PCR reactions. Bands i1 , i2 , i3 represent 6, 2, and 0.2 % of total reaction volume loaded for insert. Bands g1 , g2 , and g3 represent 6, 2 and 0.2 %, respectively, of total reaction volume loaded for genomic fragment. M 1-kb DNA ladder
    Figure Legend Snippet: Gel electrophoresis of SQ-PCR reactions. Bands i1 , i2 , i3 represent 6, 2, and 0.2 % of total reaction volume loaded for insert. Bands g1 , g2 , and g3 represent 6, 2 and 0.2 %, respectively, of total reaction volume loaded for genomic fragment. M 1-kb DNA ladder

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction

    2) Product Images from "TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha"

    Article Title: TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.001618

    Both TagF and PppA domains repress T6SS activity independently of the PpkA-mediated TssL phosphorylation pathway in A. tumefaciens . A , Phos-tag SDS-PAGE analysis to detect the phosphorylation status of TssL-His. Shown is Western blot analysis of the same volumes of Ni-NTA resins (10 μl) associated with TssL-His from different strains treated with (+) or without (−) CIAP and examined by a specific antibody against His 6 . Total protein isolated from Δ tssL was a negative control. Phos-tag SDS-PAGE revealed the upper band indicating the phosphorylated TssL-His ( p-TssL-His ) and lower band indicating unphosphorylated TssL-His. B and C , Western blot analysis of the endogenous phosphorylation status of TssL (pTssL). Shown is Western blot analysis of total proteins isolated from WT C58, Δ ppkA , Δ tssL , or C58 harboring the vector pTrc200 (V) or various overexpressing plasmids grown in AB-MES (pH 5.5) liquid culture with specific antibodies. The specific antibody for pTssL was generated against the 15-mer peptide ( 7 SSWQDLP pT VVEITEE 21 ), with phosphorylated Thr-14 of TssL underlined. RNA polymerase α subunit RpoA was an internal control. The proteins analyzed and molecular weight standards are on the left and right , respectively, and are indicated with an arrowhead when necessary. FL , full-length TagF-PppA proteins.
    Figure Legend Snippet: Both TagF and PppA domains repress T6SS activity independently of the PpkA-mediated TssL phosphorylation pathway in A. tumefaciens . A , Phos-tag SDS-PAGE analysis to detect the phosphorylation status of TssL-His. Shown is Western blot analysis of the same volumes of Ni-NTA resins (10 μl) associated with TssL-His from different strains treated with (+) or without (−) CIAP and examined by a specific antibody against His 6 . Total protein isolated from Δ tssL was a negative control. Phos-tag SDS-PAGE revealed the upper band indicating the phosphorylated TssL-His ( p-TssL-His ) and lower band indicating unphosphorylated TssL-His. B and C , Western blot analysis of the endogenous phosphorylation status of TssL (pTssL). Shown is Western blot analysis of total proteins isolated from WT C58, Δ ppkA , Δ tssL , or C58 harboring the vector pTrc200 (V) or various overexpressing plasmids grown in AB-MES (pH 5.5) liquid culture with specific antibodies. The specific antibody for pTssL was generated against the 15-mer peptide ( 7 SSWQDLP pT VVEITEE 21 ), with phosphorylated Thr-14 of TssL underlined. RNA polymerase α subunit RpoA was an internal control. The proteins analyzed and molecular weight standards are on the left and right , respectively, and are indicated with an arrowhead when necessary. FL , full-length TagF-PppA proteins.

    Techniques Used: Activity Assay, SDS Page, Western Blot, Isolation, Negative Control, Plasmid Preparation, Generated, Molecular Weight

    Conserved amino acid residues of TagF are critical for TagF–Fha interaction in A. tumefaciens . A , amino acid sequence alignment of TagF or TagF domain orthologs from selected bacterial species. Conserved amino acid residues are highlighted in black , and those used for mutagenesis are indicated with an asterisk . Sequences were aligned and highlighted by use of ClustalW2 ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). (Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.) Part of the aligned result is shown here, and the fully aligned result and full information for bacterial strains and protein accession numbers are shown in Fig. S3 A . B , Agrobacterium TagF protein is present as a monomer on gel filtration analysis in vitro . Purified His-tagged TagF domain (aa 1–214) was analyzed by SDS-PAGE. The proteins analyzed and molecular weight standards are shown on the right and left , respectively. His-tagged TagF proteins were further analyzed by use of a Superdex 75 16 × 60 column, and the elution profiles were recorded as absorbance at 280 nm showing that His-tagged TagF elutes as a single peak (∼26 kDa monomer). C , relative positions of the conserved amino acid residues in P. aeruginosa TagF Pa protein revealed as a monomer with crystal structural information according to the X-ray crystal structure of P. aeruginosa TagF monomer (Protein Data Bank entry 2QNU ). The corresponding conserved amino acid residues of A. tumefaciens TagF are indicated in parenthesis. D , yeast two-hybrid protein–protein interaction results with Fha and various TagF proteins. SD−WL medium (SD minimal medium lacking Trp and Leu) was used for selecting plasmids. SD−WLHA medium (SD minimal medium lacking Trp, Leu, His, and Ade) was used for the auxotrophic selection of bait and prey protein interactions. The positive interaction was determined by growth on SD−WLHA medium at 30 °C for at least 2 days. The positive control (+) showing interactions of SV40 large T-antigen and murine p53 and negative control (vector) are indicated.
    Figure Legend Snippet: Conserved amino acid residues of TagF are critical for TagF–Fha interaction in A. tumefaciens . A , amino acid sequence alignment of TagF or TagF domain orthologs from selected bacterial species. Conserved amino acid residues are highlighted in black , and those used for mutagenesis are indicated with an asterisk . Sequences were aligned and highlighted by use of ClustalW2 ( http://www.ebi.ac.uk/Tools/msa/clustalw2/ ). (Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.) Part of the aligned result is shown here, and the fully aligned result and full information for bacterial strains and protein accession numbers are shown in Fig. S3 A . B , Agrobacterium TagF protein is present as a monomer on gel filtration analysis in vitro . Purified His-tagged TagF domain (aa 1–214) was analyzed by SDS-PAGE. The proteins analyzed and molecular weight standards are shown on the right and left , respectively. His-tagged TagF proteins were further analyzed by use of a Superdex 75 16 × 60 column, and the elution profiles were recorded as absorbance at 280 nm showing that His-tagged TagF elutes as a single peak (∼26 kDa monomer). C , relative positions of the conserved amino acid residues in P. aeruginosa TagF Pa protein revealed as a monomer with crystal structural information according to the X-ray crystal structure of P. aeruginosa TagF monomer (Protein Data Bank entry 2QNU ). The corresponding conserved amino acid residues of A. tumefaciens TagF are indicated in parenthesis. D , yeast two-hybrid protein–protein interaction results with Fha and various TagF proteins. SD−WL medium (SD minimal medium lacking Trp and Leu) was used for selecting plasmids. SD−WLHA medium (SD minimal medium lacking Trp, Leu, His, and Ade) was used for the auxotrophic selection of bait and prey protein interactions. The positive interaction was determined by growth on SD−WLHA medium at 30 °C for at least 2 days. The positive control (+) showing interactions of SV40 large T-antigen and murine p53 and negative control (vector) are indicated.

    Techniques Used: Sequencing, Mutagenesis, Filtration, In Vitro, Purification, SDS Page, Molecular Weight, Selection, Positive Control, Negative Control, Plasmid Preparation

    3) Product Images from "Exploiting a Natural Auxotrophy for Genetic Selection"

    Article Title: Exploiting a Natural Auxotrophy for Genetic Selection

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00762-12

    Shuttle plasmid pBR103. For the DNA sequence of pBR103, see Fig. S1 in the supplemental material. repA , replication region from pFNL10; ori , replication origin from p15A; bla , β-lactamase gene; lacZ , β-galactosidase gene.
    Figure Legend Snippet: Shuttle plasmid pBR103. For the DNA sequence of pBR103, see Fig. S1 in the supplemental material. repA , replication region from pFNL10; ori , replication origin from p15A; bla , β-lactamase gene; lacZ , β-galactosidase gene.

    Techniques Used: Plasmid Preparation, Sequencing

    4) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    5) Product Images from "Transcriptome-wide identification of novel circular RNAs in soybean in response to low-phosphorus stress"

    Article Title: Transcriptome-wide identification of novel circular RNAs in soybean in response to low-phosphorus stress

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0227243

    Validation of a circRNAs (novel_circ_000237) by qPCR and Sanger sequencing. M, DL500 marker and the red arrow represent 100 bp. The yellow arrows denote the divergent primers for PCR amplification orientation.
    Figure Legend Snippet: Validation of a circRNAs (novel_circ_000237) by qPCR and Sanger sequencing. M, DL500 marker and the red arrow represent 100 bp. The yellow arrows denote the divergent primers for PCR amplification orientation.

    Techniques Used: Real-time Polymerase Chain Reaction, Sequencing, Marker, Polymerase Chain Reaction, Amplification

    6) Product Images from "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage"

    Article Title: Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

    Journal: Nature

    doi: 10.1038/nature17946

    Effects of deaminase, linker length, and linker composition on base editing a , Gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS) 3 -dCas9, and dCas9-(GGS) 3 -rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37 °C for 2 h. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. b , Coomassie-stained denaturing PAGE of the expressed and purified proteins used in (c), (d), (e), and (f). c-f , Gel-based deaminase assay showing the deamination window of base editors with deaminase–Cas9 linkers of GGS (c), (GGS) 3 (d), XTEN (e), or (GGS) 7 (f). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37 °C for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37 °C for 1 h to cleave the DNA backbone at the site of any Us. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with – if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For gel source data, see Supplementary Figure 1 .
    Figure Legend Snippet: Effects of deaminase, linker length, and linker composition on base editing a , Gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS) 3 -dCas9, and dCas9-(GGS) 3 -rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37 °C for 2 h. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. b , Coomassie-stained denaturing PAGE of the expressed and purified proteins used in (c), (d), (e), and (f). c-f , Gel-based deaminase assay showing the deamination window of base editors with deaminase–Cas9 linkers of GGS (c), (GGS) 3 (d), XTEN (e), or (GGS) 7 (f). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37 °C for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37 °C for 1 h to cleave the DNA backbone at the site of any Us. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with – if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For gel source data, see Supplementary Figure 1 .

    Techniques Used: Activity Assay, Derivative Assay, In Vitro, Incubation, Positive Control, Sequencing, Staining, Polyacrylamide Gel Electrophoresis, Purification, Isolation

    7) Product Images from "Hepatitis B virus upregulates cellular inhibitor of apoptosis protein 2 expression via the PI3K/AKT/NF-κB signaling pathway in liver cancer"

    Article Title: Hepatitis B virus upregulates cellular inhibitor of apoptosis protein 2 expression via the PI3K/AKT/NF-κB signaling pathway in liver cancer

    Journal: Oncology Letters

    doi: 10.3892/ol.2020.11267

    A NF-κB binding site in cIAP2 promoter is essential for HBV-induced cIAP2 promoter transactivation. (A) THLE-3 cells transfected with (−2,000/+55) cIAP2-Luc or pGL3-Basic and then infected with increasing doses of HBV. (B) THLE-3 cells transfected with serially truncated cIAP promoter constructs and then infected with 100 GEq/cell of HBV. Cells were lysed, and luciferase activity was measured 24 h later. (C) Predicted transcription factor binding sites within cIAP2 promoter sequence. The following transcription factor binding sites were detected in this region: 2 NFAT, 2 AP1, 3 NF-κB, 1 IRF-1 and 1 TATA box. Transcription start site is also indicated. (D) THLE-3 cells transfected with wild type or mutated (−2,000/+55)cIAP2-Luc (namely, AP1 MUT, NF-κB* MUT, NF-κB** MUT, NF-κB*** MUT and IRF-1 MUT or pGL3-Basic were infected with 100 GEq/cell of HBV. Cells were lysed, and luciferase activity was measured 24 h later. Data are presented as the mean ± standard deviation of three independent experiments. *P
    Figure Legend Snippet: A NF-κB binding site in cIAP2 promoter is essential for HBV-induced cIAP2 promoter transactivation. (A) THLE-3 cells transfected with (−2,000/+55) cIAP2-Luc or pGL3-Basic and then infected with increasing doses of HBV. (B) THLE-3 cells transfected with serially truncated cIAP promoter constructs and then infected with 100 GEq/cell of HBV. Cells were lysed, and luciferase activity was measured 24 h later. (C) Predicted transcription factor binding sites within cIAP2 promoter sequence. The following transcription factor binding sites were detected in this region: 2 NFAT, 2 AP1, 3 NF-κB, 1 IRF-1 and 1 TATA box. Transcription start site is also indicated. (D) THLE-3 cells transfected with wild type or mutated (−2,000/+55)cIAP2-Luc (namely, AP1 MUT, NF-κB* MUT, NF-κB** MUT, NF-κB*** MUT and IRF-1 MUT or pGL3-Basic were infected with 100 GEq/cell of HBV. Cells were lysed, and luciferase activity was measured 24 h later. Data are presented as the mean ± standard deviation of three independent experiments. *P

    Techniques Used: Binding Assay, Transfection, Infection, Construct, Luciferase, Activity Assay, Sequencing, Standard Deviation

    8) Product Images from "DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells"

    Article Title: DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz717

    Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.
    Figure Legend Snippet: Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.

    Techniques Used: Lysis, Methylation, Ligation, Sequencing

    9) Product Images from "Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs"

    Article Title: Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

    Journal: Genes & Development

    doi: 10.1101/gad.241950.114

    RNA-seq analysis of BioTAP-XL pull-downs. ( A ) Enrichment of repeat-derived RNA in HP1a-BioTAP cross-linked complexes from S2 cells compared with MSL3-BioTAP complexes from S2 cells detected using a random-priming approach for cDNA synthesis and Illumina
    Figure Legend Snippet: RNA-seq analysis of BioTAP-XL pull-downs. ( A ) Enrichment of repeat-derived RNA in HP1a-BioTAP cross-linked complexes from S2 cells compared with MSL3-BioTAP complexes from S2 cells detected using a random-priming approach for cDNA synthesis and Illumina

    Techniques Used: RNA Sequencing Assay, Derivative Assay

    10) Product Images from "Characterization of Transcription Factors That Regulate the Type IV Secretion System and Riboflavin Biosynthesis in Wolbachia of Brugia malayi"

    Article Title: Characterization of Transcription Factors That Regulate the Type IV Secretion System and Riboflavin Biosynthesis in Wolbachia of Brugia malayi

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051597

    w BmxR1 and w BmxR2 positively regulate the transcription of ribA lacZ reporter. β–galactosidase assays were used to measure the transcriptional activities of lacZ reporter constructs. E. coli strain C2566 transformed with the reporter plasmid and protein expression vector pET21a , or pwBmxR1 , or pwBmxR2 were tested. β–galactosidase assays were performed on induced (IPTG) and un-induced samples. Miller units are shown as mean ± standard deviations from 3 replica experiments. The ribA promoter region containing 400 bp (A) or 20 bp minimal binding sequence (B) were fused to a promoter-less lacZ and cloned into low copy plasmid pACYC184 to generate the reporter plasmid.
    Figure Legend Snippet: w BmxR1 and w BmxR2 positively regulate the transcription of ribA lacZ reporter. β–galactosidase assays were used to measure the transcriptional activities of lacZ reporter constructs. E. coli strain C2566 transformed with the reporter plasmid and protein expression vector pET21a , or pwBmxR1 , or pwBmxR2 were tested. β–galactosidase assays were performed on induced (IPTG) and un-induced samples. Miller units are shown as mean ± standard deviations from 3 replica experiments. The ribA promoter region containing 400 bp (A) or 20 bp minimal binding sequence (B) were fused to a promoter-less lacZ and cloned into low copy plasmid pACYC184 to generate the reporter plasmid.

    Techniques Used: Construct, Transformation Assay, Plasmid Preparation, Expressing, Binding Assay, Sequencing, Clone Assay

    11) Product Images from "Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution"

    Article Title: Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.885061

    Principle of the EM-seq methodology: genomic DNA can either be treated with TET2 and BGT (left) to protect both 5-mC and 5-hmC, or BGT alone (right) to protect 5-hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C).
    Figure Legend Snippet: Principle of the EM-seq methodology: genomic DNA can either be treated with TET2 and BGT (left) to protect both 5-mC and 5-hmC, or BGT alone (right) to protect 5-hmC. Subsequent deamination by APOBEC3A followed by PCR amplification allows the distinction between the unprotected substrate (read as T) from the protected cytosine derivatives (read as C).

    Techniques Used: Polymerase Chain Reaction, Amplification

    Enzymatic deamination preserves the integrity of the DNA: a. qPCR results show the quantities of undamaged amplifiable DNA templates of different sizes after the enzymatic deamination (green) and bisulfite treatments (orange and blue). All quantifications are normalized to the values obtained for the enzymatic deamination experiments. b. Agilent 2100 Bioanalyzer trace on RNA 6000 pico chip comparing equal amounts of mouse E14 genomic DNA sheared to an average of 15 kb and treated with sodium bisulfite (green), BGT and APOBEC3A (red), or TET2 and APOBEC3A (blue) over the control ssDNA (magenta). Bisulfite treatment fragmented the DNA to an average of 800 bp, while enzymatically treated DNA show no notable size differences compared to control DNA. c. Agarose gel images of end-point PCR of six amplicons ranging from 388–4229 bp illustrating upper amplicon size limit for sodium bisulfite, TET2 and APOBEC3A, or BGT and APOBEC3A treated E14 genomic DNA. d. 731 bp amplicons from the agarose gels showed in (c) were cloned, sequenced and the methylation status determined for bisulfite (left panel), enzymatically converted for 5-mC (center panel) and 5-hmC (right panel) E14 genomic DNA. Open and closed circles indicate unmethylated and methylated, respectively.
    Figure Legend Snippet: Enzymatic deamination preserves the integrity of the DNA: a. qPCR results show the quantities of undamaged amplifiable DNA templates of different sizes after the enzymatic deamination (green) and bisulfite treatments (orange and blue). All quantifications are normalized to the values obtained for the enzymatic deamination experiments. b. Agilent 2100 Bioanalyzer trace on RNA 6000 pico chip comparing equal amounts of mouse E14 genomic DNA sheared to an average of 15 kb and treated with sodium bisulfite (green), BGT and APOBEC3A (red), or TET2 and APOBEC3A (blue) over the control ssDNA (magenta). Bisulfite treatment fragmented the DNA to an average of 800 bp, while enzymatically treated DNA show no notable size differences compared to control DNA. c. Agarose gel images of end-point PCR of six amplicons ranging from 388–4229 bp illustrating upper amplicon size limit for sodium bisulfite, TET2 and APOBEC3A, or BGT and APOBEC3A treated E14 genomic DNA. d. 731 bp amplicons from the agarose gels showed in (c) were cloned, sequenced and the methylation status determined for bisulfite (left panel), enzymatically converted for 5-mC (center panel) and 5-hmC (right panel) E14 genomic DNA. Open and closed circles indicate unmethylated and methylated, respectively.

    Techniques Used: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Clone Assay, Methylation

    12) Product Images from "Complete and Draft Genome Sequences of 12 Plant-Associated Rathayibacter Strains of Known and Putative New Species"

    Article Title: Complete and Draft Genome Sequences of 12 Plant-Associated Rathayibacter Strains of Known and Putative New Species

    Journal: Microbiology Resource Announcements

    doi: 10.1128/MRA.00316-20

    Phylogenomic tree based on 20 bacterial genomes of the genus Rathayibacter . The tree is drawn to scale, with branch lengths measured in the estimated number of substitutions per site. Branch support values (rate of elementary quartets) above 0.5 are indicated at the branch points. The following newly isolated strains of seven putative new species are given in bold: (i) VKM Ac-2803 and VKM Ac-2754, (ii) VKM Ac-2759, (iii) VKM Ac-2804, (iv) VKM Ac-2760, (v) VKM Ac-2805 and VKM Ac-2762, (vi) VKM Ac-2801, and (vii) VKM Ac-2761 (the second strain of “ R. tanaceti ”). The genomic sequence of Clavibacter sepedonicus ATCC 33113 T (GenBank accession numbers AM849034.1 to AM849036.1 ) served as an outgroup (not shown).
    Figure Legend Snippet: Phylogenomic tree based on 20 bacterial genomes of the genus Rathayibacter . The tree is drawn to scale, with branch lengths measured in the estimated number of substitutions per site. Branch support values (rate of elementary quartets) above 0.5 are indicated at the branch points. The following newly isolated strains of seven putative new species are given in bold: (i) VKM Ac-2803 and VKM Ac-2754, (ii) VKM Ac-2759, (iii) VKM Ac-2804, (iv) VKM Ac-2760, (v) VKM Ac-2805 and VKM Ac-2762, (vi) VKM Ac-2801, and (vii) VKM Ac-2761 (the second strain of “ R. tanaceti ”). The genomic sequence of Clavibacter sepedonicus ATCC 33113 T (GenBank accession numbers AM849034.1 to AM849036.1 ) served as an outgroup (not shown).

    Techniques Used: Isolation, Sequencing

    13) Product Images from "Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius"

    Article Title: Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius

    Journal: Journal of Industrial Microbiology & Biotechnology

    doi: 10.1007/s10295-014-1415-6

    d -Xylose conversion in the PCP of a wild-type A. carbonarius , b mutant strain. XR xylose reductase, XDH xylitol dehydrogenase, XK xylulokinase, PPP pentose phosphate pathway
    Figure Legend Snippet: d -Xylose conversion in the PCP of a wild-type A. carbonarius , b mutant strain. XR xylose reductase, XDH xylitol dehydrogenase, XK xylulokinase, PPP pentose phosphate pathway

    Techniques Used: Mutagenesis

    14) Product Images from "Cytonuclear Coordination Is Not Immediate upon Allopolyploid Formation in Tragopogon miscellus (Asteraceae) Allopolyploids"

    Article Title: Cytonuclear Coordination Is Not Immediate upon Allopolyploid Formation in Tragopogon miscellus (Asteraceae) Allopolyploids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0144339

    Genomic (a) and cDNA (b) cleaved amplified polymorphic sequence (CAPS) results for representative samples of naturally occurring Tragopogon miscellus polyploids and the diploid parents, T . dubius (D) and T . pratensis (P). T . pratensis is the maternal parent of the short-liguled (S) individuals, and T . dubius is the maternal parent of the long-liguled (L) individuals. An asterisk (*) indicates homeolog loss in T . miscellus . The artificial hybrid contained equal mixture of T . dubius and T . pratensis genomic DNA (a) or cDNA (b). Population codes are detailed in S1 Table .
    Figure Legend Snippet: Genomic (a) and cDNA (b) cleaved amplified polymorphic sequence (CAPS) results for representative samples of naturally occurring Tragopogon miscellus polyploids and the diploid parents, T . dubius (D) and T . pratensis (P). T . pratensis is the maternal parent of the short-liguled (S) individuals, and T . dubius is the maternal parent of the long-liguled (L) individuals. An asterisk (*) indicates homeolog loss in T . miscellus . The artificial hybrid contained equal mixture of T . dubius and T . pratensis genomic DNA (a) or cDNA (b). Population codes are detailed in S1 Table .

    Techniques Used: Amplification, Sequencing

    15) Product Images from "H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast"

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast

    Journal: eLife

    doi: 10.7554/eLife.06845

    Nucleosome structure showing critical H2A residues that effect SWR1 activity. ( A ) Left : The yeast nucleosome crystal structure 1ID3 in Protein Data Bank was modeled to show histones on one face of nucleosome. Histone H2A is yellow, H2B is black and H3, H4 are gray. The domains of H2A that affect SWR1 activity-M3A (cyan), M4 (magenta), and M5 (blue) are marked. Center and right : Buried residues of histone H2A are shown by removing other histones and rotating on X-axis by 45°. ( B ) The H2A surface residue G47 in 1ID3 is shown in magenta. Bottom left : Zoom-in view shows that G47 is at the bottom of a cleft. Bottom right : Replacing Glycine for Lysine in H2A.Z histone shows the long side-chain of Lysine filling the cleft. DOI: http://dx.doi.org/10.7554/eLife.06845.006
    Figure Legend Snippet: Nucleosome structure showing critical H2A residues that effect SWR1 activity. ( A ) Left : The yeast nucleosome crystal structure 1ID3 in Protein Data Bank was modeled to show histones on one face of nucleosome. Histone H2A is yellow, H2B is black and H3, H4 are gray. The domains of H2A that affect SWR1 activity-M3A (cyan), M4 (magenta), and M5 (blue) are marked. Center and right : Buried residues of histone H2A are shown by removing other histones and rotating on X-axis by 45°. ( B ) The H2A surface residue G47 in 1ID3 is shown in magenta. Bottom left : Zoom-in view shows that G47 is at the bottom of a cleft. Bottom right : Replacing Glycine for Lysine in H2A.Z histone shows the long side-chain of Lysine filling the cleft. DOI: http://dx.doi.org/10.7554/eLife.06845.006

    Techniques Used: Activity Assay

    Nucleosomal histone and DNA elements critical for SWR1 activity and model for SWR1-mediated H2A-H2B displacement. ( A ) Yeast nucleosome structure PDB 1ID3 was modeled to show one face of the nucleosome and the histone-fold elements that are critical for SWR1 activation. The SWR1 footprint is shown in blue. The gap-sensitive region, 17–22 nt from dyad, is shown in cyan. Residues of H2A that affect SWR1 activity are shown in magenta. ( B ) Nucleosome model showing histone-DNA and histone–histone interactions that hold H2A-H2B within the nucleosome. Also shown is the gap-sensitive region, where SWR1 interacts with nucleosome DNA leading to eviction of H2A/H2B and concomitant deposition of H2A.Z/H2B. DOI: http://dx.doi.org/10.7554/eLife.06845.011
    Figure Legend Snippet: Nucleosomal histone and DNA elements critical for SWR1 activity and model for SWR1-mediated H2A-H2B displacement. ( A ) Yeast nucleosome structure PDB 1ID3 was modeled to show one face of the nucleosome and the histone-fold elements that are critical for SWR1 activation. The SWR1 footprint is shown in blue. The gap-sensitive region, 17–22 nt from dyad, is shown in cyan. Residues of H2A that affect SWR1 activity are shown in magenta. ( B ) Nucleosome model showing histone-DNA and histone–histone interactions that hold H2A-H2B within the nucleosome. Also shown is the gap-sensitive region, where SWR1 interacts with nucleosome DNA leading to eviction of H2A/H2B and concomitant deposition of H2A.Z/H2B. DOI: http://dx.doi.org/10.7554/eLife.06845.011

    Techniques Used: Activity Assay, Activation Assay

    Position of SWR1 footprint on linker-distal face of nucleosome. The 601 DNA-containing nucleosome structure PDB 3MVD was modeled to highlight the position of the SWR1 footprint in blue on the linker-distal side of the dyad axis. The H2A on the linker-distal face is in yellow. DOI: http://dx.doi.org/10.7554/eLife.06845.008
    Figure Legend Snippet: Position of SWR1 footprint on linker-distal face of nucleosome. The 601 DNA-containing nucleosome structure PDB 3MVD was modeled to highlight the position of the SWR1 footprint in blue on the linker-distal side of the dyad axis. The H2A on the linker-distal face is in yellow. DOI: http://dx.doi.org/10.7554/eLife.06845.008

    Techniques Used:

    SWR1 mediates histone exchange without net change of nucleosome position. ( A ) Left : EMSA (6% native PAGE) shows INO80-mediated nucleosome sliding. An asymmetrically positioned 601 nucleosome with a 43 bp and 0 bp DNA linker was used for the sliding assay. Right : SWR1-mediated incorporation of H2A.Z-H2B dimer (without 3FLAG epitope tag). Incorporation of H2A.Z in nucleosome was confirmed by immunoblotting with anti-H2A.Z antibody. ( B ) Hydroxyl radical footprinting strategy. A canonical nucleosome with 60 bp and 0 bp linker DNA and fluorescence end-label (bottom strand) was used as substrate for histone replacement, followed by hydroxyl radical treatment and separation by 6% native PAGE. ( C ) Recovered DNA from gel slices containing AA, AZ, and ZZ states was analyzed on DNA sequencing gels. ( D ) Intensity plots for AA, AZ, and ZZ nucleosomes. DOI: http://dx.doi.org/10.7554/eLife.06845.012
    Figure Legend Snippet: SWR1 mediates histone exchange without net change of nucleosome position. ( A ) Left : EMSA (6% native PAGE) shows INO80-mediated nucleosome sliding. An asymmetrically positioned 601 nucleosome with a 43 bp and 0 bp DNA linker was used for the sliding assay. Right : SWR1-mediated incorporation of H2A.Z-H2B dimer (without 3FLAG epitope tag). Incorporation of H2A.Z in nucleosome was confirmed by immunoblotting with anti-H2A.Z antibody. ( B ) Hydroxyl radical footprinting strategy. A canonical nucleosome with 60 bp and 0 bp linker DNA and fluorescence end-label (bottom strand) was used as substrate for histone replacement, followed by hydroxyl radical treatment and separation by 6% native PAGE. ( C ) Recovered DNA from gel slices containing AA, AZ, and ZZ states was analyzed on DNA sequencing gels. ( D ) Intensity plots for AA, AZ, and ZZ nucleosomes. DOI: http://dx.doi.org/10.7554/eLife.06845.012

    Techniques Used: Clear Native PAGE, Footprinting, Fluorescence, DNA Sequencing

    SWR1 binding to nucleosome core particle with gaps on both sides of dyad. Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles. DOI: http://dx.doi.org/10.7554/eLife.06845.010
    Figure Legend Snippet: SWR1 binding to nucleosome core particle with gaps on both sides of dyad. Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles. DOI: http://dx.doi.org/10.7554/eLife.06845.010

    Techniques Used: Binding Assay, Labeling, Agarose Gel Electrophoresis

    SWR1 binding to nucleosome core particles containing H2A or H2A.Z histone. EMSA shows SWR1 binding to Alexa 647-labeled H2A- and H2A.Z-nucleosome core particles (1 nM). Free and bound complexes are resolved on 1.3% agarose gel. Bottom: binding curves for H2A- and H2A.Z-nucleosome core particles. DOI: http://dx.doi.org/10.7554/eLife.06845.004
    Figure Legend Snippet: SWR1 binding to nucleosome core particles containing H2A or H2A.Z histone. EMSA shows SWR1 binding to Alexa 647-labeled H2A- and H2A.Z-nucleosome core particles (1 nM). Free and bound complexes are resolved on 1.3% agarose gel. Bottom: binding curves for H2A- and H2A.Z-nucleosome core particles. DOI: http://dx.doi.org/10.7554/eLife.06845.004

    Techniques Used: Binding Assay, Labeling, Agarose Gel Electrophoresis

    16) Product Images from "Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects"

    Article Title: Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1090

    ( A ) Proposed mechanism of Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE). Chromosomal mutations are introduced via single-stranded oligonucleotide recombination on the lagging strand of replicating DNA, mediated by the β subunit of λ Red recombinase. Under ordinary conditions (left), the newly-synthesized strand is hemimethylated, which enables the MMR system, composed of MutS, MutL and MutH, to bind to mismatched lesions and restore the wild-type allele. In standard MAGE (middle), the MMR system is eliminated, allowing incorporation of the mismatched base and its complement base into the genome of one daughter cell after a second round of replication (red). However, permanent disabling of mismatch repair also allows the accumulation of off-target mutations (yellow) during subsequent manipulations. In TM-MAGE (right), transient overexpression of DNA adenine methylase (Dam) is hypothesized to generate significant methylation of the newly-synthesized strand, disabling detection of incorporated mismatches by the MMR system and reducing the rate of off-target mutations that accumulate in subsequent manipulations as Dam expression returns to its baseline level. ( B ) Schematic of TM-MAGE system plasmids pMA7 (top) and pMA7SacB (bottom). Plasmid pMA7 contains an artificial operon of the gene encoding the β subunit of λ Red recombinase and dam controlled by an arabinose-inducible (P BAD ) promoter. Plasmid pMA7SacB additionally contains constitutively expressed sacB (levansucrase from B. subtilis ) to allow for plasmid curing by sucrose counterselection.
    Figure Legend Snippet: ( A ) Proposed mechanism of Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE). Chromosomal mutations are introduced via single-stranded oligonucleotide recombination on the lagging strand of replicating DNA, mediated by the β subunit of λ Red recombinase. Under ordinary conditions (left), the newly-synthesized strand is hemimethylated, which enables the MMR system, composed of MutS, MutL and MutH, to bind to mismatched lesions and restore the wild-type allele. In standard MAGE (middle), the MMR system is eliminated, allowing incorporation of the mismatched base and its complement base into the genome of one daughter cell after a second round of replication (red). However, permanent disabling of mismatch repair also allows the accumulation of off-target mutations (yellow) during subsequent manipulations. In TM-MAGE (right), transient overexpression of DNA adenine methylase (Dam) is hypothesized to generate significant methylation of the newly-synthesized strand, disabling detection of incorporated mismatches by the MMR system and reducing the rate of off-target mutations that accumulate in subsequent manipulations as Dam expression returns to its baseline level. ( B ) Schematic of TM-MAGE system plasmids pMA7 (top) and pMA7SacB (bottom). Plasmid pMA7 contains an artificial operon of the gene encoding the β subunit of λ Red recombinase and dam controlled by an arabinose-inducible (P BAD ) promoter. Plasmid pMA7SacB additionally contains constitutively expressed sacB (levansucrase from B. subtilis ) to allow for plasmid curing by sucrose counterselection.

    Techniques Used: Multiplex Assay, Synthesized, Over Expression, Methylation, Expressing, Plasmid Preparation

    Transient expression of Dam enables efficient recombineering. ARFs for xylA Y13* in strains EcNR2, K-12 MG1655/pMA7 and K-12 MG1655/pMA7SacB following one cycle of MAGE with a 90 bp phosphorothioated oligonucleotide. ARFs were determined both by counting red (wild-type allele) and white (mutant allele) colonies appearing following plating on MacConkey agar containing 1% xylose, and by amplicon sequencing within the xylA locus. Error bars indicated standard deviations about the mean of three replicates (two for K-12 MG1655/pMA7SacB).
    Figure Legend Snippet: Transient expression of Dam enables efficient recombineering. ARFs for xylA Y13* in strains EcNR2, K-12 MG1655/pMA7 and K-12 MG1655/pMA7SacB following one cycle of MAGE with a 90 bp phosphorothioated oligonucleotide. ARFs were determined both by counting red (wild-type allele) and white (mutant allele) colonies appearing following plating on MacConkey agar containing 1% xylose, and by amplicon sequencing within the xylA locus. Error bars indicated standard deviations about the mean of three replicates (two for K-12 MG1655/pMA7SacB).

    Techniques Used: Expressing, Mutagenesis, Amplification, Sequencing

    17) Product Images from "Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications"

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsw038

    Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.
    Figure Legend Snippet: Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.

    Techniques Used: Amplification, Construct

    18) Product Images from "Modulation of ALDH5A1 and SLC22A7 by microRNA hsa-miR-29a-3p in human liver cells"

    Article Title: Modulation of ALDH5A1 and SLC22A7 by microRNA hsa-miR-29a-3p in human liver cells

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2015.09.020

    The hsa-miR-29a-3p oligonucleotides directly interacted with (A) ABCC6 , (B) ALDH5A1 , and (C) SLC22A7 mRNA oligonucleotides in vitro *The reagents were used in RNA EMSA containing ABCC6 , ALDH5A1 , and SLC22A7 mRNA oligonucleotides, under the same experimental conditions. NC, nonspecific competitor. Lanes 1 and 2 indicated the mobility of each type of oligonucleotide; lane 3 indicated the mobility status of the miRNA:mRNA complex formed by the interaction of hsa-miR-29a-3p oligonucleotides with ABCC6 , ALDH5A1 , or SLC22A7 mRNA oligonucleotides; lanes 4 and 5 revealed the mobility shift status of miRNA:mRNA complex in the presence of excess unlabeled nonspecific competitors and specific competitors (hsa-miR-29a-3p). Lane 6 showed complexes formed using the cytoplasmic extracts from HepaRG cells incubated with hsa-miR-29a-3p oligonucleotides and ABCC6 , ALDH5A1 , and SLC22A7 mRNA oligonucleotides. Lanes 7 and 8 showed the mobility shift status of protein: miRNA:mRNA complexes in the presence of excess unlabeled nonspecific competitors and specific hsa-miR-29a-3p competitors. Lanes 9 and 10 indicated the mobility status of miRNA/mRNA/protein complexes with Ago1 or Ago2 antibodies. Arrows (left) indicate the oligonucleotide complexes in lane 3. Arrows (right) indicate the miRNA/mRNA/protein complexes formed by oligonucleotides and cytoplasmic proteins in lanes 6–10 in (B) and (C). SS indicates the supershift complex formed by the miRNA/mRNA/protein and antibody against Ago1 in lane 9 in (B). (D and E) Histogram corresponds to the relative densitometry quantification of the key miRNA/mRNA complexes or miRNA/mRNA/protein complexes observed in (A, B or C), compared to those in lane 3 or lane 6, respectively. ** P
    Figure Legend Snippet: The hsa-miR-29a-3p oligonucleotides directly interacted with (A) ABCC6 , (B) ALDH5A1 , and (C) SLC22A7 mRNA oligonucleotides in vitro *The reagents were used in RNA EMSA containing ABCC6 , ALDH5A1 , and SLC22A7 mRNA oligonucleotides, under the same experimental conditions. NC, nonspecific competitor. Lanes 1 and 2 indicated the mobility of each type of oligonucleotide; lane 3 indicated the mobility status of the miRNA:mRNA complex formed by the interaction of hsa-miR-29a-3p oligonucleotides with ABCC6 , ALDH5A1 , or SLC22A7 mRNA oligonucleotides; lanes 4 and 5 revealed the mobility shift status of miRNA:mRNA complex in the presence of excess unlabeled nonspecific competitors and specific competitors (hsa-miR-29a-3p). Lane 6 showed complexes formed using the cytoplasmic extracts from HepaRG cells incubated with hsa-miR-29a-3p oligonucleotides and ABCC6 , ALDH5A1 , and SLC22A7 mRNA oligonucleotides. Lanes 7 and 8 showed the mobility shift status of protein: miRNA:mRNA complexes in the presence of excess unlabeled nonspecific competitors and specific hsa-miR-29a-3p competitors. Lanes 9 and 10 indicated the mobility status of miRNA/mRNA/protein complexes with Ago1 or Ago2 antibodies. Arrows (left) indicate the oligonucleotide complexes in lane 3. Arrows (right) indicate the miRNA/mRNA/protein complexes formed by oligonucleotides and cytoplasmic proteins in lanes 6–10 in (B) and (C). SS indicates the supershift complex formed by the miRNA/mRNA/protein and antibody against Ago1 in lane 9 in (B). (D and E) Histogram corresponds to the relative densitometry quantification of the key miRNA/mRNA complexes or miRNA/mRNA/protein complexes observed in (A, B or C), compared to those in lane 3 or lane 6, respectively. ** P

    Techniques Used: In Vitro, Mobility Shift, Incubation

    hsa-miR-29a-3p inhibited luciferase reporter gene expression controlled by the 3′-UTRs of ALDH5A1 or SLC22A7 (A) Free energy analyses for the interactions between hsa-miR-29a-3p and the targeting sequences or site-mutants present the in ALDH5A1 3′-UTR, or SLC22A7 3′-UTR. Δ G , free energy; the underlined letter, the mutated base. (B) ALDH5A1-CU and ALDH5A1-Mut plasmids or (C) SLC22A7-CU and SLC22A7-Mut plasmids were transiently transfected into 293T and HepG2 cells, together with 50 nmol/L hsa-miR-29a-3p mimic or miRNA negative control. Cells were harvested 48 h after transfection. Three independent experiments, each in triplicate, were performed, and fold changes of luciferase activity were calculated by defining the activity of ALDH5A1-CU plasmid, or SLC22A7-CU, together with miRNA negative control as unity. ** P
    Figure Legend Snippet: hsa-miR-29a-3p inhibited luciferase reporter gene expression controlled by the 3′-UTRs of ALDH5A1 or SLC22A7 (A) Free energy analyses for the interactions between hsa-miR-29a-3p and the targeting sequences or site-mutants present the in ALDH5A1 3′-UTR, or SLC22A7 3′-UTR. Δ G , free energy; the underlined letter, the mutated base. (B) ALDH5A1-CU and ALDH5A1-Mut plasmids or (C) SLC22A7-CU and SLC22A7-Mut plasmids were transiently transfected into 293T and HepG2 cells, together with 50 nmol/L hsa-miR-29a-3p mimic or miRNA negative control. Cells were harvested 48 h after transfection. Three independent experiments, each in triplicate, were performed, and fold changes of luciferase activity were calculated by defining the activity of ALDH5A1-CU plasmid, or SLC22A7-CU, together with miRNA negative control as unity. ** P

    Techniques Used: Luciferase, Expressing, Transfection, Negative Control, Activity Assay, Plasmid Preparation

    miRNA hsa-miR-29a-3p inhibited endogenous ALDH5A1 and SLC22A7 expression in HepaRG cells Differentiated HepaRG cells were transiently transfected using 50 nmol/L miRNA negative control or hsa-miR-29a-3p mimic. Each assay was carried out in triplicate. * P
    Figure Legend Snippet: miRNA hsa-miR-29a-3p inhibited endogenous ALDH5A1 and SLC22A7 expression in HepaRG cells Differentiated HepaRG cells were transiently transfected using 50 nmol/L miRNA negative control or hsa-miR-29a-3p mimic. Each assay was carried out in triplicate. * P

    Techniques Used: Expressing, Transfection, Negative Control

    Chemical compounds NSC-156306 and NSC-642957 down-regulated ALDH5A1 and SLC22A7 expression in HepaRG cells Differentiated HepaRG cells were treated with 0, 10, or 100 nmol/L NSC-156306 or NSC-642957. Cells were harvested 48 h after treatments. Each assay was done at least 3 times. * P
    Figure Legend Snippet: Chemical compounds NSC-156306 and NSC-642957 down-regulated ALDH5A1 and SLC22A7 expression in HepaRG cells Differentiated HepaRG cells were treated with 0, 10, or 100 nmol/L NSC-156306 or NSC-642957. Cells were harvested 48 h after treatments. Each assay was done at least 3 times. * P

    Techniques Used: Expressing

    19) Product Images from "Destabilization of B2 RNA by EZH2 activates the stress response"

    Article Title: Destabilization of B2 RNA by EZH2 activates the stress response

    Journal: Cell

    doi: 10.1016/j.cell.2016.11.041

    EZH2 triggers cleavage of B2 RNA in vitro A) B2 sub-family consensus sequences. B) Incubation (22°C, 13h) of B2 RNA (200 nM) with EZH2 (25 nM) results in B2 cleavage. C) Incubation with 25 nM control proteins, GST and EED, does not result in significant cutting (22°C, 13h). D) Cleaved RNA fragments (asterisks) are subjected to deep sequencing (x-axis: start coordinates for the sequenced reads). E) Incubation of in vitro-transcribed RNAs (100 nM) with EZH2 (50 nM) results in cleavage only of B2. RNAs were mixed with EZH2 and incubated at 37°C or 4°C for 30 min. B2 was also incubated with FLAG peptide (50 nM) at 37°C as control. F) Kinetic analysis of B2 cleavage in the presence of EZH2. 25 nM EZH2 was incubated with 200 nM B2 RNA (37°C for 0–100 min.) and run on a 6% TBE-Urea-PAGE. G) Fraction of full-length B2 RNA at each time point from panel E (arrow) was plotted as a function of time (two independent experiments). Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope is the observed cleavage rate constant ( k obs ). H) Table of calculated ob k obs and RNA half-lives for B2 in the presence of various test proteins. I) Rate of B2 cleavage depends on the concentration of EZH2 protein. 50 nM B2 RNA is incubated with increasing concentrations of EZH2 in vitro (37°C, 20min) and run on a 6% TBE-Urea PAGE. J) Kinetic analysis showing that the rate of B2 cleavage depends on the concentration of EZH2 (two independent experiments). 200 nM B2 RNA is incubated with increasing EZH2 concentrations (25–500 nM) at 37°C and the amount of remaining full-length B2 RNA is plotted as a function of time. Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope approximated observed rate constant ( k obs ). K) k obs values from panel I are plotted as a function of EZH2 concentration. Arrowhead, full-length B2 RNA. Asterisks, cleaved B2 fragments. High R 2 , excellent fit of datapoints to the curve.
    Figure Legend Snippet: EZH2 triggers cleavage of B2 RNA in vitro A) B2 sub-family consensus sequences. B) Incubation (22°C, 13h) of B2 RNA (200 nM) with EZH2 (25 nM) results in B2 cleavage. C) Incubation with 25 nM control proteins, GST and EED, does not result in significant cutting (22°C, 13h). D) Cleaved RNA fragments (asterisks) are subjected to deep sequencing (x-axis: start coordinates for the sequenced reads). E) Incubation of in vitro-transcribed RNAs (100 nM) with EZH2 (50 nM) results in cleavage only of B2. RNAs were mixed with EZH2 and incubated at 37°C or 4°C for 30 min. B2 was also incubated with FLAG peptide (50 nM) at 37°C as control. F) Kinetic analysis of B2 cleavage in the presence of EZH2. 25 nM EZH2 was incubated with 200 nM B2 RNA (37°C for 0–100 min.) and run on a 6% TBE-Urea-PAGE. G) Fraction of full-length B2 RNA at each time point from panel E (arrow) was plotted as a function of time (two independent experiments). Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope is the observed cleavage rate constant ( k obs ). H) Table of calculated ob k obs and RNA half-lives for B2 in the presence of various test proteins. I) Rate of B2 cleavage depends on the concentration of EZH2 protein. 50 nM B2 RNA is incubated with increasing concentrations of EZH2 in vitro (37°C, 20min) and run on a 6% TBE-Urea PAGE. J) Kinetic analysis showing that the rate of B2 cleavage depends on the concentration of EZH2 (two independent experiments). 200 nM B2 RNA is incubated with increasing EZH2 concentrations (25–500 nM) at 37°C and the amount of remaining full-length B2 RNA is plotted as a function of time. Cleavage rate constants were then determined by a linear fit using the differential form of the rate equation for an irreversible, first-order reaction. The slope approximated observed rate constant ( k obs ). K) k obs values from panel I are plotted as a function of EZH2 concentration. Arrowhead, full-length B2 RNA. Asterisks, cleaved B2 fragments. High R 2 , excellent fit of datapoints to the curve.

    Techniques Used: In Vitro, Incubation, Sequencing, Polyacrylamide Gel Electrophoresis, Concentration Assay

    20) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    21) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
    Figure Legend Snippet: Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue

    Techniques Used: Flow Cytometry, In Vitro, Incubation, Recombinant, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Molecular Weight, Marker, Amplification

    22) Product Images from "DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells"

    Article Title: DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz717

    Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.
    Figure Legend Snippet: Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.

    Techniques Used: Lysis, Methylation, Ligation, Sequencing

    23) Product Images from "FANCM and FAAP24 Maintain Genome Stability via Cooperative as well as Unique Functions"

    Article Title: FANCM and FAAP24 Maintain Genome Stability via Cooperative as well as Unique Functions

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2012.12.010

    FANCM deletion leads to defective recombination-independent repair of ICLs ( A ) Schematics of the reporter reactivation-based recombination-independent ICL repair assay. Control and crosslinked reporter represent the unmodified and site-specifically crosslinked plasmid substrates, respectively. ( B ) Recombination-independent ICL repair efficiencies in FANCM and FAAP24 mutant cell lines. Error bars represent standard deviation of 6 independent experiments (* P
    Figure Legend Snippet: FANCM deletion leads to defective recombination-independent repair of ICLs ( A ) Schematics of the reporter reactivation-based recombination-independent ICL repair assay. Control and crosslinked reporter represent the unmodified and site-specifically crosslinked plasmid substrates, respectively. ( B ) Recombination-independent ICL repair efficiencies in FANCM and FAAP24 mutant cell lines. Error bars represent standard deviation of 6 independent experiments (* P

    Techniques Used: Plasmid Preparation, Mutagenesis, Standard Deviation

    Homologous replacement targeting of FANCM and FAAP24 loci in HCT-116 cells ( A ) Left panel, schematics of FANCM targeting strategy. Right panel, PCR genotyping of FANCM −/− . ( B ) Left panel, schematics of FAAP24 targeting strategy. Right panel, PCR genotyping of FAAP24 −/− . Numbered boxes indicate exons. Light shaded boxes indicate exon(s) targeted for deletion. Targeted allele (n) depicts the initial targeting event where the targeted exon(s) is replaced by the Neo R cassette. Knockout allele (−) refers to the Cre-treated targeted allele which resulted in the removal of the Neo R cassette. ( C ) Western blot detecting FANCM and FAAP24 protein loss in somatic knockout cell lines. The asterisk (*) marks a nonspecific band recognized by the FAAP24 antibody. ( D ) Immunoblot detecting MMC-induced monoubiquitination of FANCD2 in FANCM −/− cells complemented with wild-type Flag-FANCM. ( E ) Immunoblot detecting MMC-induced monoubiquitination of FANCD2 in FAAP24 −/− cells complemented with wild-type Flag-FAAP24. L and S represent monoubiquitinated and native forms of FANCD2, respectively.
    Figure Legend Snippet: Homologous replacement targeting of FANCM and FAAP24 loci in HCT-116 cells ( A ) Left panel, schematics of FANCM targeting strategy. Right panel, PCR genotyping of FANCM −/− . ( B ) Left panel, schematics of FAAP24 targeting strategy. Right panel, PCR genotyping of FAAP24 −/− . Numbered boxes indicate exons. Light shaded boxes indicate exon(s) targeted for deletion. Targeted allele (n) depicts the initial targeting event where the targeted exon(s) is replaced by the Neo R cassette. Knockout allele (−) refers to the Cre-treated targeted allele which resulted in the removal of the Neo R cassette. ( C ) Western blot detecting FANCM and FAAP24 protein loss in somatic knockout cell lines. The asterisk (*) marks a nonspecific band recognized by the FAAP24 antibody. ( D ) Immunoblot detecting MMC-induced monoubiquitination of FANCD2 in FANCM −/− cells complemented with wild-type Flag-FANCM. ( E ) Immunoblot detecting MMC-induced monoubiquitination of FANCD2 in FAAP24 −/− cells complemented with wild-type Flag-FAAP24. L and S represent monoubiquitinated and native forms of FANCD2, respectively.

    Techniques Used: Polymerase Chain Reaction, Knock-Out, Western Blot

    Differential cellular sensitivities and chromosomal abnormalities in FANCM and FAAP24 single and double mutants ( A ) Clonogenic survival of FANCM −/− and FAAP24 −/− mutants treated with MMC. Error-bars represent standard deviations from six independent experiments with triplicated plates. ( B ) Clonogenic survival of FANCM −/− and FAAP24 −/− mutants treated with cisplatin. Error-bars represent standard deviations from four independent experiments with triplicated plates. ( C ) Chromosome breakage in FANCM −/− and FAAP24 −/− mutant cells exposed to MMC (40 ng/ml for 18 hr). Arrows indicate visible chromosome breaks. ( D ) Quantification of chromosomal abnormality in FANCM −/− and FAAP24 −/− mutant cells. 40 metaphase spreads were scored for each cell line. Bars represent average chromosomal breakage per spread. ( E ) MMC-induced radial chromosomes in FANCM −/− and FAAP24 −/− mutant cells. Quantifications of radial chromosomes in 80 metaphase spreads with each genotype were shown. Error-bars depict standard deviations.
    Figure Legend Snippet: Differential cellular sensitivities and chromosomal abnormalities in FANCM and FAAP24 single and double mutants ( A ) Clonogenic survival of FANCM −/− and FAAP24 −/− mutants treated with MMC. Error-bars represent standard deviations from six independent experiments with triplicated plates. ( B ) Clonogenic survival of FANCM −/− and FAAP24 −/− mutants treated with cisplatin. Error-bars represent standard deviations from four independent experiments with triplicated plates. ( C ) Chromosome breakage in FANCM −/− and FAAP24 −/− mutant cells exposed to MMC (40 ng/ml for 18 hr). Arrows indicate visible chromosome breaks. ( D ) Quantification of chromosomal abnormality in FANCM −/− and FAAP24 −/− mutant cells. 40 metaphase spreads were scored for each cell line. Bars represent average chromosomal breakage per spread. ( E ) MMC-induced radial chromosomes in FANCM −/− and FAAP24 −/− mutant cells. Quantifications of radial chromosomes in 80 metaphase spreads with each genotype were shown. Error-bars depict standard deviations.

    Techniques Used: Mutagenesis

    FANCM and FAAP24 coordinately promote the activation of Fanconi anemia pathway ( A–C ) Immunoblots detecting FANCD2 monoubiquitination in cells with indicated genotypes, treated or mock-treated with MMC (200 ng/ml, 16 hr). ( D ) Formation of MMC-induced (500 ng/ml) FANCD2 nuclear foci in FANCM and FAAP24 mutants. ( E ) Formation of MMC-induced FANCD2 nuclear foci in complemented FANCM −/− and FAAP24 −/− cells. ( F ) Quantification of FANCD2 foci in FANCM and FAAP24 mutants and their isogenic complements. Data represent three independent experiments and error bars depict standard deviation derived from 5 data sets. ( G ) Chromatin association of FANCM, FAAP24, FANCA, FANCG, FANCL and FANCD2 in response to MMC treatment (200 ng/ml, 6 hr).
    Figure Legend Snippet: FANCM and FAAP24 coordinately promote the activation of Fanconi anemia pathway ( A–C ) Immunoblots detecting FANCD2 monoubiquitination in cells with indicated genotypes, treated or mock-treated with MMC (200 ng/ml, 16 hr). ( D ) Formation of MMC-induced (500 ng/ml) FANCD2 nuclear foci in FANCM and FAAP24 mutants. ( E ) Formation of MMC-induced FANCD2 nuclear foci in complemented FANCM −/− and FAAP24 −/− cells. ( F ) Quantification of FANCD2 foci in FANCM and FAAP24 mutants and their isogenic complements. Data represent three independent experiments and error bars depict standard deviation derived from 5 data sets. ( G ) Chromatin association of FANCM, FAAP24, FANCA, FANCG, FANCL and FANCD2 in response to MMC treatment (200 ng/ml, 6 hr).

    Techniques Used: Activation Assay, Western Blot, Standard Deviation, Derivative Assay

    FANCM and FAAP24 suppress sister chromatid exchanges ( A ) Representative metaphase chromosome spreads showing MMC-induced SCEs (arrows) in FANCM and FAAP24 mutants. ( B ) Quantification of basal level (mock-treated) and MMC-induced (20 ng/ml for 18 hr) SCEs in FANCM and FAAP24 mutants. 30 metaphases were scored for each sample and bars represent averages. The asterisks (*) denote P
    Figure Legend Snippet: FANCM and FAAP24 suppress sister chromatid exchanges ( A ) Representative metaphase chromosome spreads showing MMC-induced SCEs (arrows) in FANCM and FAAP24 mutants. ( B ) Quantification of basal level (mock-treated) and MMC-induced (20 ng/ml for 18 hr) SCEs in FANCM and FAAP24 mutants. 30 metaphases were scored for each sample and bars represent averages. The asterisks (*) denote P

    Techniques Used:

    FAAP24 is required for ATR-mediated checkpoint signaling with distinct damage specificity ( A ) Ser317 phosphorylation of Chk1 in FANCM and FAAP24 mutant cells exposed to MMC (150 ng/ml) and harvested at indicated time points. ( B ) Ser317 phosphorylation of Chk1 in FANCM and FAAP24 mutant cells exposed to UV (20 J/m 2 ) and harvested at indicated time points. ( C ) Ser317 phosphorylation of Chk1 in FANCM and FAAP24 mutant cells exposed to HU (1 mM) and harvested at indicated time points. Chk1 and β-Actin serve as loading controls. ( D ) Co-immunoprecipitation of wild-type FAAP24 and the FAAP24-V198A mutant with FANCM. ( E ) MMC-induced Chk1 activation in FAAP24 −/− cells complemented with Flag-FAAP24-wild type or Flag-FAAP24-V198A mutant. ( F ) Clonogenic survival of FAAP24 −/− cells complemented with Flag-FAAP24-wild type or Flag-FAAP24-V198A. Error-bars represent standard deviations from three independent experiments with triplicated plates.
    Figure Legend Snippet: FAAP24 is required for ATR-mediated checkpoint signaling with distinct damage specificity ( A ) Ser317 phosphorylation of Chk1 in FANCM and FAAP24 mutant cells exposed to MMC (150 ng/ml) and harvested at indicated time points. ( B ) Ser317 phosphorylation of Chk1 in FANCM and FAAP24 mutant cells exposed to UV (20 J/m 2 ) and harvested at indicated time points. ( C ) Ser317 phosphorylation of Chk1 in FANCM and FAAP24 mutant cells exposed to HU (1 mM) and harvested at indicated time points. Chk1 and β-Actin serve as loading controls. ( D ) Co-immunoprecipitation of wild-type FAAP24 and the FAAP24-V198A mutant with FANCM. ( E ) MMC-induced Chk1 activation in FAAP24 −/− cells complemented with Flag-FAAP24-wild type or Flag-FAAP24-V198A mutant. ( F ) Clonogenic survival of FAAP24 −/− cells complemented with Flag-FAAP24-wild type or Flag-FAAP24-V198A. Error-bars represent standard deviations from three independent experiments with triplicated plates.

    Techniques Used: Mutagenesis, Immunoprecipitation, Activation Assay

    24) Product Images from "Controlled expression of functional miR-122 with a ligand inducible expression system"

    Article Title: Controlled expression of functional miR-122 with a ligand inducible expression system

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-10-76

    Short hairpins based on miR-122 designed to express a particular siRNA guide strand . An siRNA sequence used previously to silence firefly luciferase (FLuc) was placed in the miR-122 stem-loop structure as shown schematically. a. Top: The sequence of the FLuc target mRNA (shown 3' to 5') was used to extend the complementary siRNA guide strand (middle underlined). Bottom: Schematic representation of the two short hairpins designed to express firefly luciferase siRNA. The 19 nt sequence of the FLuc siRNA guide strand is underlined. The sequence of the miRNA expected from these short hairpins is 23 nt in length (black). FLuc-ShM contains a mismatch (magenta). Sequence residues originating from the miR-122 pre-miRNA are blue. All hairpin forms are designed to produce identical guide strand sequences. b. Inducible silencing of firefly luciferase. FLuc short hairpin constructs were transiently transfected into NIH3T3-47 cells along with the Fluc reporter plasmid pGL3Luc and pCMV-lacZ as a transfection control. Cells were treated with 0.5 μM RSL1 or DMSO for 48 h, and cell lysates were subsequently assayed for firefly luciferase and β-galactosidase for normalization. The luciferase activity remaining 48 h after induction is shown as a percent of activity from matched transfected non-induced cells. Results represent the mean (+/- SD) of 3 experiments. p-values represent comparison of percent remaining activity for FLuc short hairpins vs. non-targeting control miR-122 short hairpins. * p
    Figure Legend Snippet: Short hairpins based on miR-122 designed to express a particular siRNA guide strand . An siRNA sequence used previously to silence firefly luciferase (FLuc) was placed in the miR-122 stem-loop structure as shown schematically. a. Top: The sequence of the FLuc target mRNA (shown 3' to 5') was used to extend the complementary siRNA guide strand (middle underlined). Bottom: Schematic representation of the two short hairpins designed to express firefly luciferase siRNA. The 19 nt sequence of the FLuc siRNA guide strand is underlined. The sequence of the miRNA expected from these short hairpins is 23 nt in length (black). FLuc-ShM contains a mismatch (magenta). Sequence residues originating from the miR-122 pre-miRNA are blue. All hairpin forms are designed to produce identical guide strand sequences. b. Inducible silencing of firefly luciferase. FLuc short hairpin constructs were transiently transfected into NIH3T3-47 cells along with the Fluc reporter plasmid pGL3Luc and pCMV-lacZ as a transfection control. Cells were treated with 0.5 μM RSL1 or DMSO for 48 h, and cell lysates were subsequently assayed for firefly luciferase and β-galactosidase for normalization. The luciferase activity remaining 48 h after induction is shown as a percent of activity from matched transfected non-induced cells. Results represent the mean (+/- SD) of 3 experiments. p-values represent comparison of percent remaining activity for FLuc short hairpins vs. non-targeting control miR-122 short hairpins. * p

    Techniques Used: Sequencing, Luciferase, Construct, Transfection, Plasmid Preparation, Activity Assay

    25) Product Images from "Controlled expression of functional miR-122 with a ligand inducible expression system"

    Article Title: Controlled expression of functional miR-122 with a ligand inducible expression system

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-10-76

    Knock down of target genes by induced expression of miR-122 . a. Glycogen synthase-3' UTR targeted by miR-122. NIH3T3-47/X1-miR122 cells were transfected with reporter plasmids pTKGLuc (control), or pTKGLuc-GYS with the (glycogen synthase 3' untranslated region (UTR) and mIR-122 target sites schematic, top panel). The luciferase activity remaining 48 h after induction is plotted as a percent of activity from control cells. (*p = 0.0255; Error bar = -/+1SD) (See Methods for 3' UTR sequence coordinates.) b. Western blot analysis of aldolase A, an endogenous target of miR-122,. NIH3T3-47/X1-miR122 cells were treated with DMSO or 0.5 μM RSL1, then cell lysates were used for western blot analysis. The sample of 9 days post treatment is shown. Aldolase A protein quantification was calculated after LiCor scanning of Western blot normalized for loading with alpha-beta tubulin. A single miR-122 target site in aldo A is located at position 27-34 in the aldoA 3' UTR (top panel).
    Figure Legend Snippet: Knock down of target genes by induced expression of miR-122 . a. Glycogen synthase-3' UTR targeted by miR-122. NIH3T3-47/X1-miR122 cells were transfected with reporter plasmids pTKGLuc (control), or pTKGLuc-GYS with the (glycogen synthase 3' untranslated region (UTR) and mIR-122 target sites schematic, top panel). The luciferase activity remaining 48 h after induction is plotted as a percent of activity from control cells. (*p = 0.0255; Error bar = -/+1SD) (See Methods for 3' UTR sequence coordinates.) b. Western blot analysis of aldolase A, an endogenous target of miR-122,. NIH3T3-47/X1-miR122 cells were treated with DMSO or 0.5 μM RSL1, then cell lysates were used for western blot analysis. The sample of 9 days post treatment is shown. Aldolase A protein quantification was calculated after LiCor scanning of Western blot normalized for loading with alpha-beta tubulin. A single miR-122 target site in aldo A is located at position 27-34 in the aldoA 3' UTR (top panel).

    Techniques Used: Expressing, Transfection, Luciferase, Activity Assay, Sequencing, Western Blot

    26) Product Images from "LIN-9 Phosphorylation on Threonine-96 Is Required for Transcriptional Activation of LIN-9 Target Genes and Promotes Cell Cycle Progression"

    Article Title: LIN-9 Phosphorylation on Threonine-96 Is Required for Transcriptional Activation of LIN-9 Target Genes and Promotes Cell Cycle Progression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0087620

    cyclin E is essential for phosphorylation of Thr-96 in LIN-9 in vivo in primary cells. (A) p-LIN-9 Thr-96 antibody is specific. 293T cells were cotransfected with plasmids encoding GFP-LIN-9, FLAG-Cdk3 and cyclin E, and cell extracts were subjected to immunoprecipitation using a monoclonal LIN-9 antibody. IP material was split into two, and incubated for 1 hour at 30°C with or without lambda phosphatase. Samples were subjected to Western Blot using our p-LIN-9 Thr-96 antibody (upper panel). Equal loading of LIN-9 was confirmed by incubating the same membrane with a rabbit antibody against LIN-9 (lower panel). (B) HUVECs were transfected with siRNA control or siRNA directed against Cyclin E1 (Santa Cruz) using Dharmafect 4 (Dharmacon) and harvested after 48 hours. Western Blots was performed with the indicated antibodies. (C) T98G cells were starved in serum-free medium for 36 hours followed by the addition of growth medium (supplemented with 10% serum). Cells were harvested by tripsynization at the indicated time after the addition of serum and use for Western blot or FACS analysis. Western blots were performed as described in Methods.
    Figure Legend Snippet: cyclin E is essential for phosphorylation of Thr-96 in LIN-9 in vivo in primary cells. (A) p-LIN-9 Thr-96 antibody is specific. 293T cells were cotransfected with plasmids encoding GFP-LIN-9, FLAG-Cdk3 and cyclin E, and cell extracts were subjected to immunoprecipitation using a monoclonal LIN-9 antibody. IP material was split into two, and incubated for 1 hour at 30°C with or without lambda phosphatase. Samples were subjected to Western Blot using our p-LIN-9 Thr-96 antibody (upper panel). Equal loading of LIN-9 was confirmed by incubating the same membrane with a rabbit antibody against LIN-9 (lower panel). (B) HUVECs were transfected with siRNA control or siRNA directed against Cyclin E1 (Santa Cruz) using Dharmafect 4 (Dharmacon) and harvested after 48 hours. Western Blots was performed with the indicated antibodies. (C) T98G cells were starved in serum-free medium for 36 hours followed by the addition of growth medium (supplemented with 10% serum). Cells were harvested by tripsynization at the indicated time after the addition of serum and use for Western blot or FACS analysis. Western blots were performed as described in Methods.

    Techniques Used: In Vivo, Immunoprecipitation, Incubation, Western Blot, Transfection, FACS

    27) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
    Figure Legend Snippet: Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue

    Techniques Used: Flow Cytometry, In Vitro, Incubation, Recombinant, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Molecular Weight, Marker, Amplification

    28) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
    Figure Legend Snippet: Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue

    Techniques Used: Flow Cytometry, In Vitro, Incubation, Recombinant, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Molecular Weight, Marker, Amplification

    29) Product Images from "Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells"

    Article Title: Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells

    Journal: Respiratory Research

    doi: 10.1186/s12931-018-0861-5

    Orthogonal validation of RNA-Seq data. a-d Expression of select genes was validated by qPCR; ESR1 ( a ), Connective tissue growth factor ( CTGF, b ), VIM ( c ), and Matrix metalloproteinase 2 ( MMP2 , d ), in an identical and independent experiment. Bars represent expression [Log2(Fold Change)] of each gene in the RNA-Seq analysis, and black dots represent expression [Log2(Fold Change)] in each sample ( n = 6) in the orthogonal experiment as determined by qPCR relative to vehicle control (DMSO). Target gene expression as measured by qPCR was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Asterisks (*) indicate differential expression compared to controls [Log2(Fold Change) ≥ |0.6| and FDR-corrected p -value
    Figure Legend Snippet: Orthogonal validation of RNA-Seq data. a-d Expression of select genes was validated by qPCR; ESR1 ( a ), Connective tissue growth factor ( CTGF, b ), VIM ( c ), and Matrix metalloproteinase 2 ( MMP2 , d ), in an identical and independent experiment. Bars represent expression [Log2(Fold Change)] of each gene in the RNA-Seq analysis, and black dots represent expression [Log2(Fold Change)] in each sample ( n = 6) in the orthogonal experiment as determined by qPCR relative to vehicle control (DMSO). Target gene expression as measured by qPCR was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Asterisks (*) indicate differential expression compared to controls [Log2(Fold Change) ≥ |0.6| and FDR-corrected p -value

    Techniques Used: RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction

    30) Product Images from "Simultaneous and stoichiometric purification of hundreds of oligonucleotides"

    Article Title: Simultaneous and stoichiometric purification of hundreds of oligonucleotides

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04870-w

    Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized
    Figure Legend Snippet: Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized

    Techniques Used: Purification, Synthesized, Sequencing, Concentration Assay, Oligo Synthesis, Magnetic Beads, Next-Generation Sequencing, Standard Deviation, Binding Assay

    31) Product Images from "Protein Synthesis Using A Reconstituted Cell-Free System"

    Article Title: Protein Synthesis Using A Reconstituted Cell-Free System

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    doi: 10.1002/0471142727.mb1631s108

    SDS-PAGE analysis of the reverse purification of the DHFR control protein synthesized in the PURExpress reaction. M: molecular weight standards (kDa); Lane 1: Control PURExpress reaction with no input template, Lane 2: PURExpress reaction with the DHFR
    Figure Legend Snippet: SDS-PAGE analysis of the reverse purification of the DHFR control protein synthesized in the PURExpress reaction. M: molecular weight standards (kDa); Lane 1: Control PURExpress reaction with no input template, Lane 2: PURExpress reaction with the DHFR

    Techniques Used: SDS Page, Purification, Synthesized, Molecular Weight

    Scanned image of a SDS-PAGE gel of proteins synthesized in the PURExpress reactions and labeled with FluoroTect™ Green Lys . Lane 1: DHFR; lane 2: GFP; lane 3: Renilla luciferase; lane 4: Firefly luciferase; lane 5: E. coli β-galactosidase.
    Figure Legend Snippet: Scanned image of a SDS-PAGE gel of proteins synthesized in the PURExpress reactions and labeled with FluoroTect™ Green Lys . Lane 1: DHFR; lane 2: GFP; lane 3: Renilla luciferase; lane 4: Firefly luciferase; lane 5: E. coli β-galactosidase.

    Techniques Used: SDS Page, Synthesized, Labeling, Luciferase

    Coomassie blue-stained SDS-PAGE gel of a panel of proteins synthesized in the PUREpxress reactions. Molecular weight standards (kDa) are shown on the left. Red dots indicate the expected positions (bands) of synthesized proteins. Lane 1, dihydrofolate
    Figure Legend Snippet: Coomassie blue-stained SDS-PAGE gel of a panel of proteins synthesized in the PUREpxress reactions. Molecular weight standards (kDa) are shown on the left. Red dots indicate the expected positions (bands) of synthesized proteins. Lane 1, dihydrofolate

    Techniques Used: Staining, SDS Page, Synthesized, Molecular Weight

    32) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
    Figure Legend Snippet: Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue

    Techniques Used: Flow Cytometry, In Vitro, Incubation, Recombinant, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Molecular Weight, Marker, Amplification

    33) Product Images from "Analysis of 3D genomic interactions identifies candidate host genes that transposable elements potentially regulate"

    Article Title: Analysis of 3D genomic interactions identifies candidate host genes that transposable elements potentially regulate

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1598-7

    ERV interactions are constrained by the different levels of nuclear organization a Whole chromosome view of 4Tran-PCR signal for RLTR4 integrations on chromosomes 3 and 8. The single integrations shown for these chromosomes are highlighted with an arrow over the plot and the regions identified as significantly interacting with these sites are shown under 4Tran-PCR signal as boxes. Hi-C data is represented by the PC-score calculated for each 50 kb bin. A positive PC score is characteristic of A regions, while a negative score is associate with B regions. b Violin plots representing the PC score for all regions identified as interacting in cis with the RLTR4 integration in chromosomes 3 and 8. An integration in compartment A leads to contacts with other compartment A regions, while the reverse is true for an integration in compartment B on chromosome 8. c , d ” section
    Figure Legend Snippet: ERV interactions are constrained by the different levels of nuclear organization a Whole chromosome view of 4Tran-PCR signal for RLTR4 integrations on chromosomes 3 and 8. The single integrations shown for these chromosomes are highlighted with an arrow over the plot and the regions identified as significantly interacting with these sites are shown under 4Tran-PCR signal as boxes. Hi-C data is represented by the PC-score calculated for each 50 kb bin. A positive PC score is characteristic of A regions, while a negative score is associate with B regions. b Violin plots representing the PC score for all regions identified as interacting in cis with the RLTR4 integration in chromosomes 3 and 8. An integration in compartment A leads to contacts with other compartment A regions, while the reverse is true for an integration in compartment B on chromosome 8. c , d ” section

    Techniques Used: Polymerase Chain Reaction, Hi-C

    34) Product Images from "Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq"

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    Journal: Nature Communications

    doi: 10.1038/ncomms15335

    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.
    Figure Legend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Techniques Used: Mutagenesis, Two Tailed Test

    35) Product Images from "Simultaneous and stoichiometric purification of hundreds of oligonucleotides"

    Article Title: Simultaneous and stoichiometric purification of hundreds of oligonucleotides

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04870-w

    Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized
    Figure Legend Snippet: Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized

    Techniques Used: Purification, Synthesized, Sequencing, Concentration Assay, Oligo Synthesis, Magnetic Beads, Next-Generation Sequencing, Standard Deviation, Binding Assay

    36) Product Images from "Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs"

    Article Title: Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

    Journal: Genes & Development

    doi: 10.1101/gad.241950.114

    RNA-seq analysis of BioTAP-XL pull-downs. ( A ) Enrichment of repeat-derived RNA in HP1a-BioTAP cross-linked complexes from S2 cells compared with MSL3-BioTAP complexes from S2 cells detected using a random-priming approach for cDNA synthesis and Illumina
    Figure Legend Snippet: RNA-seq analysis of BioTAP-XL pull-downs. ( A ) Enrichment of repeat-derived RNA in HP1a-BioTAP cross-linked complexes from S2 cells compared with MSL3-BioTAP complexes from S2 cells detected using a random-priming approach for cDNA synthesis and Illumina

    Techniques Used: RNA Sequencing Assay, Derivative Assay

    37) Product Images from "A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors"

    Article Title: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.117960

    Regeneration of ribozymes after cleavage enables selection and an NGS-based assay that are correlated with in vivo activity. a. Both CleaveSeq and DRIVER use the same core transcription (in the presence or absence of ligand(s)) and regeneration method of the 5’ cleaved product after cleavage. b . The regeneration method selectively restores the 5’ cleaved portion of the ribozyme and replaces the prefix sequence in the input library with a new prefix (e.g., “W” prefix is replaced with “Z” prefix for cleaved RNA molecules). The process starts with co-transcriptional cleavage of a DNA template library (i.e., simultaneous RNA transcription and cleavage of the product RNA in the presence or absence of ligand(s) to form a population of RNA molecules some of which have undergone self-cleavage thereby removing part of the ribozyme and the fixed prefix sequence from their 5’-end. The resulting pool of RNA is mixed with an oligonucleotide ( Z-Splint ; Dataset S2) that anneals to the 3’-end of the RNA for reverse transcription. The oligonucleotide subsequently hybridizes to the nascent cDNA, forming a partially self-annealing double-stranded hairpin structure that brings together the ends of molecules derived from the cleaved RNA, which is self-ligated with high efficiency. The resulting circularized ligation product is cut at two uracil locations included in the oligonucleotide by Uracil-Specific Excision Reagent (USER), to release a linear DNA strand harboring the desired sequence with a new prefix sequence. Two distinct populations of DNA molecules result: those corresponding to RNA that did not cleave and retain the template-determined prefix sequence and those corresponding to cleaved RNA that have the new prefix. The resulting DNA is selectively PCR-amplified with primers that extend the product with either the T7 promoter (for DRIVER) or NGS adapters (for CleaveSeq). c. Using the regeneration method, the CleaveSeq assay measures the relative abundance of molecules that underwent cleavage or not to provide estimates of cleavage fractions and switching (fold change of cleavage with the addition of ligand) for each sequence in an input library. d. Top panel, representative comparison of computed cleavage fractions for two replicates independently carried through the CleaveSeq assay from transcription through NGS analysis (N=12,025, at least 100 reads/sequence in each analysis). Bottom panel, the same data plotted with the ratio of the two replicate cleavage fraction measurements on the x-axis and the standard error of the ratio on the y-axis. Significant (p
    Figure Legend Snippet: Regeneration of ribozymes after cleavage enables selection and an NGS-based assay that are correlated with in vivo activity. a. Both CleaveSeq and DRIVER use the same core transcription (in the presence or absence of ligand(s)) and regeneration method of the 5’ cleaved product after cleavage. b . The regeneration method selectively restores the 5’ cleaved portion of the ribozyme and replaces the prefix sequence in the input library with a new prefix (e.g., “W” prefix is replaced with “Z” prefix for cleaved RNA molecules). The process starts with co-transcriptional cleavage of a DNA template library (i.e., simultaneous RNA transcription and cleavage of the product RNA in the presence or absence of ligand(s) to form a population of RNA molecules some of which have undergone self-cleavage thereby removing part of the ribozyme and the fixed prefix sequence from their 5’-end. The resulting pool of RNA is mixed with an oligonucleotide ( Z-Splint ; Dataset S2) that anneals to the 3’-end of the RNA for reverse transcription. The oligonucleotide subsequently hybridizes to the nascent cDNA, forming a partially self-annealing double-stranded hairpin structure that brings together the ends of molecules derived from the cleaved RNA, which is self-ligated with high efficiency. The resulting circularized ligation product is cut at two uracil locations included in the oligonucleotide by Uracil-Specific Excision Reagent (USER), to release a linear DNA strand harboring the desired sequence with a new prefix sequence. Two distinct populations of DNA molecules result: those corresponding to RNA that did not cleave and retain the template-determined prefix sequence and those corresponding to cleaved RNA that have the new prefix. The resulting DNA is selectively PCR-amplified with primers that extend the product with either the T7 promoter (for DRIVER) or NGS adapters (for CleaveSeq). c. Using the regeneration method, the CleaveSeq assay measures the relative abundance of molecules that underwent cleavage or not to provide estimates of cleavage fractions and switching (fold change of cleavage with the addition of ligand) for each sequence in an input library. d. Top panel, representative comparison of computed cleavage fractions for two replicates independently carried through the CleaveSeq assay from transcription through NGS analysis (N=12,025, at least 100 reads/sequence in each analysis). Bottom panel, the same data plotted with the ratio of the two replicate cleavage fraction measurements on the x-axis and the standard error of the ratio on the y-axis. Significant (p

    Techniques Used: Selection, Next-Generation Sequencing, In Vivo, Activity Assay, Sequencing, Derivative Assay, Ligation, Polymerase Chain Reaction, Amplification

    38) Product Images from "A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries"

    Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

    Journal: Journal of Advanced Research

    doi: 10.1016/j.jare.2019.12.005

    Transcriptomic comparison of the RNA-seq libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols. Human total RNA (1.0 µg) was subjected to the rRNA removal process by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis (in replicate). ( A ) The average valid reads (RPKM) for the RTR2D ( a ) and the NEB kit ( b ) are depicted in various categories of transcripts. ( B ) Scatter plots and correlations of mRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a b ). ( C ) Scatter plots and correlations of lncRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a b ).
    Figure Legend Snippet: Transcriptomic comparison of the RNA-seq libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols. Human total RNA (1.0 µg) was subjected to the rRNA removal process by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis (in replicate). ( A ) The average valid reads (RPKM) for the RTR2D ( a ) and the NEB kit ( b ) are depicted in various categories of transcripts. ( B ) Scatter plots and correlations of mRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a b ). ( C ) Scatter plots and correlations of lncRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a b ).

    Techniques Used: RNA Sequencing Assay, Next-Generation Sequencing

    Comparison of human rRNA removal specificity and efficiency between the RTR2D procedure and NEBNext® rRNA Depletion kit. Human total RNA (1.0 µg) was subjected to the RTR2D (R2D) (with the pooled rRNA probes) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p
    Figure Legend Snippet: Comparison of human rRNA removal specificity and efficiency between the RTR2D procedure and NEBNext® rRNA Depletion kit. Human total RNA (1.0 µg) was subjected to the RTR2D (R2D) (with the pooled rRNA probes) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p

    Techniques Used:

    Comparison of mouse rRNA removal specificity and efficiency between the RTR2D procedure and NEB Next rRNA Depletion kit. Mouse total RNA (1.0 µg) was subjected to the RTR2D (R2D) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p
    Figure Legend Snippet: Comparison of mouse rRNA removal specificity and efficiency between the RTR2D procedure and NEB Next rRNA Depletion kit. Mouse total RNA (1.0 µg) was subjected to the RTR2D (R2D) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p

    Techniques Used:

    Nutlin3A-induced transcriptomic changes determined by RNA-seq analysis of the NGS libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols . Exponentially growing human osteosarcoma line SJSA1 cells were treated with 2 µM Nutlin3A or DMSO control for 24 h and subjected to total RNA isolation. Human total RNA (1.0 µg) was subjected to rRNA removal by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis. ( A ) Scatter plots of mRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Venn diagrams were generated by using more stringent criteria for the differentially expressed transcripts for up-regulated ( c ) (total relative reads > 54) and down-regulated transcripts ( d ) (total relative reads > 13). ( B ) Scatter plots of lncRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Significant up and down-regulated transcripts were defined as log2FoldChange > 1 and
    Figure Legend Snippet: Nutlin3A-induced transcriptomic changes determined by RNA-seq analysis of the NGS libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols . Exponentially growing human osteosarcoma line SJSA1 cells were treated with 2 µM Nutlin3A or DMSO control for 24 h and subjected to total RNA isolation. Human total RNA (1.0 µg) was subjected to rRNA removal by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis. ( A ) Scatter plots of mRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Venn diagrams were generated by using more stringent criteria for the differentially expressed transcripts for up-regulated ( c ) (total relative reads > 54) and down-regulated transcripts ( d ) (total relative reads > 13). ( B ) Scatter plots of lncRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Significant up and down-regulated transcripts were defined as log2FoldChange > 1 and

    Techniques Used: RNA Sequencing Assay, Next-Generation Sequencing, Isolation, Generated

    39) Product Images from "Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway"

    Article Title: Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    Journal: Scientific Reports

    doi: 10.1038/srep08572

    Targeted integration into Mgat1 locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in Mgat1 locus. (b) Indel frequency in Mgat1 locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNAs. The genomic regions covering sgRNA target sites were amplified, then subjected to Miseq analysis. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. (c) Agarose gel of 5′/3′ junction PCR on transiently transfected cells and stable cell pools. (++) Stable cells expressing both mCherry and Zsgreen1-DR; (+-) stable cells expressing only mCherry. (d) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pools were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Population analysis of Mgat1 disrupted cells by F-RCA-I staining. Based on red/green fluorescence intensity of stable cell pools, which was further selected with RCA-I or not, alteration of fluorescence intensity was analyzed upon F-RCA-I staining. Each scatter plot was divided by four quadrants, denoted by Q1 to Q4. Q3 and Q4 populations, marked by red squares, represent negative stained cells with F-RCA-I, indicating functional knockout of Mgat1 locus. Numerals below the red squares show the percentage of Q3 and Q4. (f) Agarose gel of out-out PCR results of stable pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (2.0 kb for sgRNA1 target site; 1.9 kb for sgRNA5 target site) or targeted integration (5.6 kb for sgRNA1 target site; 5.5 kb for sgRNA5 target site). (g) Relative copy number of Mgat1 and mCherry regions in clonal cells, as described in Fig. 1(e) . ( Top ) sgRNA1 target site ( Bottom ) sgRNA5 target site.
    Figure Legend Snippet: Targeted integration into Mgat1 locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in Mgat1 locus. (b) Indel frequency in Mgat1 locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNAs. The genomic regions covering sgRNA target sites were amplified, then subjected to Miseq analysis. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. (c) Agarose gel of 5′/3′ junction PCR on transiently transfected cells and stable cell pools. (++) Stable cells expressing both mCherry and Zsgreen1-DR; (+-) stable cells expressing only mCherry. (d) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pools were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Population analysis of Mgat1 disrupted cells by F-RCA-I staining. Based on red/green fluorescence intensity of stable cell pools, which was further selected with RCA-I or not, alteration of fluorescence intensity was analyzed upon F-RCA-I staining. Each scatter plot was divided by four quadrants, denoted by Q1 to Q4. Q3 and Q4 populations, marked by red squares, represent negative stained cells with F-RCA-I, indicating functional knockout of Mgat1 locus. Numerals below the red squares show the percentage of Q3 and Q4. (f) Agarose gel of out-out PCR results of stable pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (2.0 kb for sgRNA1 target site; 1.9 kb for sgRNA5 target site) or targeted integration (5.6 kb for sgRNA1 target site; 5.5 kb for sgRNA5 target site). (g) Relative copy number of Mgat1 and mCherry regions in clonal cells, as described in Fig. 1(e) . ( Top ) sgRNA1 target site ( Bottom ) sgRNA5 target site.

    Techniques Used: CRISPR, Sequencing, Transfection, Expressing, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Stable Transfection, Purification, Staining, Fluorescence, Functional Assay, Knock-Out

    Targeted integration into COSMC locus using CRISPR/Cas9. (a) Schematic illustration of the targeting strategy for the specific locus of interest. Donor plasmid consists of three parts: short homology arms flanking sgRNA target site cleaved by Cas9 (red triangle), mCherry and neomycin resistance gene expression cassettes inside homology arms, and ZsGreen1-DR expression cassette outside homology arms. Upon DSBs induced by CRISPR/Cas9, HDR-mediated repair can be used to insert a total size of 3.7 kb of expression cassettes through recombination of the target locus with donor plasmids. Primer position for 5′/3′ junction PCR is indicated. (b) Agarose gel of 5′/3′ junction PCR on transiently transfected cells and stable cell pools. An asterisk indicates the use of linearized donor plasmid. M, 1 kb DNA ladder (c) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. The chromatogram sequence of junction PCR amplicon was compared with the reference sequence at the genome-donor boundaries. (d) Agarose gel of out-out PCR results of stable cell pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (1.6 kb) or targeted integration (5.3 kb). (e) Relative copy number of COSMC and mCherry regions in clonal cells. Each plot shows the relative copy number of each region in comparison to the reference sample. Genomic DNA of wild type CHO-S and Clone #1 and was used as the reference for COSMC and mCherry region, respectively (shown in red). The error bars represent the standard deviations (n ≥ 3).
    Figure Legend Snippet: Targeted integration into COSMC locus using CRISPR/Cas9. (a) Schematic illustration of the targeting strategy for the specific locus of interest. Donor plasmid consists of three parts: short homology arms flanking sgRNA target site cleaved by Cas9 (red triangle), mCherry and neomycin resistance gene expression cassettes inside homology arms, and ZsGreen1-DR expression cassette outside homology arms. Upon DSBs induced by CRISPR/Cas9, HDR-mediated repair can be used to insert a total size of 3.7 kb of expression cassettes through recombination of the target locus with donor plasmids. Primer position for 5′/3′ junction PCR is indicated. (b) Agarose gel of 5′/3′ junction PCR on transiently transfected cells and stable cell pools. An asterisk indicates the use of linearized donor plasmid. M, 1 kb DNA ladder (c) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. The chromatogram sequence of junction PCR amplicon was compared with the reference sequence at the genome-donor boundaries. (d) Agarose gel of out-out PCR results of stable cell pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (1.6 kb) or targeted integration (5.3 kb). (e) Relative copy number of COSMC and mCherry regions in clonal cells. Each plot shows the relative copy number of each region in comparison to the reference sample. Genomic DNA of wild type CHO-S and Clone #1 and was used as the reference for COSMC and mCherry region, respectively (shown in red). The error bars represent the standard deviations (n ≥ 3).

    Techniques Used: CRISPR, Plasmid Preparation, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Stable Transfection, Sequencing, Purification, Amplification

    Targeted integration into LdhA locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in LdhA locus. (b) Indel frequency in LdhA locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNA. The genomic regions covering sgRNA target sites were amplified, then subjected to deep sequencing analysis using Miseq. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. Investigation of target specific knock-in in transiently transfected and stable cell pools analyzed by (c) Agarose gel of 5′/3′ junction PCR (d) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Relative copy number of LdhA and mCherry regions in clonal cells, as described in Fig. 1(e) .
    Figure Legend Snippet: Targeted integration into LdhA locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in LdhA locus. (b) Indel frequency in LdhA locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNA. The genomic regions covering sgRNA target sites were amplified, then subjected to deep sequencing analysis using Miseq. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. Investigation of target specific knock-in in transiently transfected and stable cell pools analyzed by (c) Agarose gel of 5′/3′ junction PCR (d) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Relative copy number of LdhA and mCherry regions in clonal cells, as described in Fig. 1(e) .

    Techniques Used: CRISPR, Sequencing, Transfection, Expressing, Amplification, Plasmid Preparation, Knock-In, Stable Transfection, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Purification

    40) Product Images from "Multiplex single-molecule interaction profiling of DNA barcoded proteins"

    Article Title: Multiplex single-molecule interaction profiling of DNA barcoded proteins

    Journal: Nature

    doi: 10.1038/nature13761

    Covalent immobilization of barcoding DNAs is required for in situ polony amplification Representative images of polonies amplified from barcoding DNA templates without ( a ) or with ( b ) 5’-acrydite modifications. Oversized polonies or polony clusters shown in ( a ) were resulted from template-drifting-induced multiple seeding events during the amplification.
    Figure Legend Snippet: Covalent immobilization of barcoding DNAs is required for in situ polony amplification Representative images of polonies amplified from barcoding DNA templates without ( a ) or with ( b ) 5’-acrydite modifications. Oversized polonies or polony clusters shown in ( a ) were resulted from template-drifting-induced multiple seeding events during the amplification.

    Techniques Used: In Situ, Amplification

    Polony quantification of various barcoded proteins a, Plot showing the average number of polonies detected at a single imaging position vs. the average number of barcoding DNA templates predicted by real-time PCR quantification. Data represent mean values of 100 measurements; error bars, 95% CL. b, Log–log plot of total numbers of polonies detected vs. dilution factors. Data represent mean values of two technical replicates.
    Figure Legend Snippet: Polony quantification of various barcoded proteins a, Plot showing the average number of polonies detected at a single imaging position vs. the average number of barcoding DNA templates predicted by real-time PCR quantification. Data represent mean values of 100 measurements; error bars, 95% CL. b, Log–log plot of total numbers of polonies detected vs. dilution factors. Data represent mean values of two technical replicates.

    Techniques Used: Imaging, Real-time Polymerase Chain Reaction

    Related Articles

    Ligation:

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    Magnetic Beads:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Lambda DNA Preparation:

    Article Title: Global DNA methylation in old subjects is correlated with frailty
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    SYBR Green Assay:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Incubation:

    Article Title: Global DNA methylation in old subjects is correlated with frailty
    Article Snippet: .. One hundred nanograms of methylated and unmethylated lambda DNA were separately incubated with 5 U of HpaII and MspI restriction endonucleases (New England Biolabs) at 37°C overnight and successively at 65°C for 20 min to inactivate the endonucleases. .. The samples were loaded on a 1.4% agarose gel, electrophoresed in TAE (Tris/Acetate/EDTA) buffer and stained with ethidium bromide.

    Article Title: Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner
    Article Snippet: .. The DNA was incubated with restriction enzymes MspI and HpaII for at least 3 h (restriction enzymes MspI and HpaII (New England Biolabs) differ in sensitivity to cytosine methylation). .. The genomic DNA digested by the restriction enzymes and undigested DNA were used as templates for the real-time PCR reaction.

    Methylation:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Article Title: Global DNA methylation in old subjects is correlated with frailty
    Article Snippet: .. One hundred nanograms of methylated and unmethylated lambda DNA were separately incubated with 5 U of HpaII and MspI restriction endonucleases (New England Biolabs) at 37°C overnight and successively at 65°C for 20 min to inactivate the endonucleases. .. The samples were loaded on a 1.4% agarose gel, electrophoresed in TAE (Tris/Acetate/EDTA) buffer and stained with ethidium bromide.

    Article Title: Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner
    Article Snippet: .. The DNA was incubated with restriction enzymes MspI and HpaII for at least 3 h (restriction enzymes MspI and HpaII (New England Biolabs) differ in sensitivity to cytosine methylation). .. The genomic DNA digested by the restriction enzymes and undigested DNA were used as templates for the real-time PCR reaction.

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function
    Article Snippet: .. Approximately 5 μg of genomic DNA was digested overnight at 37°C with 20 U of methylation-sensitive restriction enzyme HpaII (New England Biolabs, R0171S) or 40 U of methylation insensitive restriction enzyme MspI (New England Biolabs, R0106S). .. The reaction buffer included Cut Smart buffer 1 × (New England Biolabs), spermidine 0.1 M, DTT 0.1 M and 0.25 μl of RNaseH in 50 μl final volume.

    Sequencing:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

    Plasmid Preparation:

    Article Title: Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles
    Article Snippet: .. Reagents The reagents and materials used in this study were purchased from the manufacturers indicated in parentheses: CpG methyltransferase (M.Sss I), T4 DNA ligase, and restriction enzymes Hpa II, Msp I, Sbf I, and Stu I (New England Biolabs, MA, USA) it guarantees that the efficiency of their restriction enzymes is almost and the methylation of CpG blocks 100% Hpa II digestion reaction; EpiTect Bisulfite Kit and AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany); Oligonucleotides (Operon, Alameda, CA, USA); Magnetic beads coated with streptavidin (Dynabeads® M-280 Streptavidin) (Dynal, Oslo, Norway); TITANIUM Taq DNA polymerase (Takara Bio, Kusatsu, Japan); GenElute™ Agarose Spin Columns (Sigma-Aldrich, St. Louis, MO, USA); Ligation Convenience Kit (Nippon Gene, Tokyo, Japan); pGEM® -T Easy Vector (Promega, Madison, WI, USA); Competent Cell DH5α and Insert Check-Ready (Toyobo, Osaka, Japan); LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany); POP-7™ Polymer, GeneScan™ 500 LIZ® Size Standard, and BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific Inc., San Diego, CA, USA). .. Animals and tissues Thirteen-week old male C57BL/6 J mice (n = 3) purchased from CLEA Japan Inc. (CLEA Japan Inc., Tokyo, Japan) were sacrificed by cervical dislocation to collect liver, kidney, and hippocampus samples.

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    New England Biolabs user enzyme
    Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; <t>HSI_insert_1</t> construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without <t>USER</t> treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.
    User Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

    doi: 10.1093/dnares/dsw038

    Figure Lengend Snippet: Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.

    Article Snippet: Treatments with the USER enzyme were performed on HSI_insert_1 by incubating 2 × 107 copies HSI_insert construct with 1 U USER enzyme (NEB) in 1× Phusion HF Buffer in a reaction volume of 20 µl at 37 °C for 30 min prior to amplification.

    Techniques: Amplification, Construct

    Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized

    Journal: Nature Communications

    Article Title: Simultaneous and stoichiometric purification of hundreds of oligonucleotides

    doi: 10.1038/s41467-018-04870-w

    Figure Lengend Snippet: Stoichiometrically normalizing oligonucleotide purification (SNOP) concept and workflow. a The input reagents for SNOP are chemically synthesized oligonucleotide precursors P 1 through P N that contain imperfect synthesis products with 5′ truncations and/or internal deletions, and with potentially very different concentrations. SNOP produces a pool of oligonucleotide products O 1 through O N that has high fractions of oligos with perfect sequence, and with all products at roughly equal concentration. SNOP uses a single biotinylated capture probe oligonucleotide synthesized with a degenerate “SWSWSW” randomer subsequence. Each instance of the randomer is complementary to one precursor tag sequence. The different instances of the capture probe are all at roughly equal concentration, due to split-pool oligo synthesis. Precursors with perfect tag sequences hybridize to the probe and are captured by streptavidin-coated magnetic beads. Subsequent cleavage at the deoxyuracil (dU) site using the USER enzyme mix ( https://www.neb.com/products/m5505-user-enzyme ) releases the oligo products into solution. Setting the capture probe to be the limiting reagent allows all SNOP products to be all at roughly equal concentrations. b SNOP enriches the fraction of perfect oligos because synthesis errors are correlated; molecules with no truncations or deletions in the tag sequences are also more likely to not have any deletions in the oligo product sequence. Shown in this panel are NGS sequence analysis results of a pool of N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos (see Methods for library preparation details). c SNOP is very sensitive to small sequence changes in the tag; even single-nucleotide variations result in significantly reduced binding yield (see also Supplementary Note). This property allows SNOP products to be both highly pure and stoichiometrically normalized

    Article Snippet: Subsequent solid-phase separation using streptavidin-coated magnetic beads removes unbound precursors, and applying USER enzyme mix (New England Biolabs) cleaves the oligo products from the tags at the dU site.

    Techniques: Purification, Synthesized, Sequencing, Concentration Assay, Oligo Synthesis, Magnetic Beads, Next-Generation Sequencing, Standard Deviation, Binding Assay

    Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are

    Journal: Nature chemical biology

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity

    doi: 10.1038/nchembio.1793

    Figure Lengend Snippet: Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are

    Article Snippet: Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously .

    Techniques: Modification, High Throughput Screening Assay, DNA Sequencing, Amplification, Transfection, Expressing, Plasmid Preparation

    Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. ( a ) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. ( b ) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. ( c ) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.

    Journal: Nature chemical biology

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity

    doi: 10.1038/nchembio.1793

    Figure Lengend Snippet: Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. ( a ) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. ( b ) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. ( c ) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.

    Article Snippet: Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously .

    Techniques: Binding Assay, Activity Assay, Standard Deviation

    Design of primers for USER cloning with pMS26. Two choices of translation signal in the 5′-UTR are shown. The gene-specific sequence illustrated is of fnuDIIM . The underlined 21 bp of longer translation signal (LTS) is from the tacp regulatory region ( 34 ) ; the short signal (STS) is a truncation of it. The LTS and downstream primers illustrated are the same as primers 5 and 6 of Table 4 ; the STS construct was made but not used in this report. Fusion of lacZ to the signal as shown creates an RBS/ATG spacing of six, within the usual range of spacing ( 35 ); lacZ native spacing is seven ( 36 ).

    Journal: Nucleic Acids Research

    Article Title: A versatile element for gene addition in bacterial chromosomes

    doi: 10.1093/nar/gkr1085

    Figure Lengend Snippet: Design of primers for USER cloning with pMS26. Two choices of translation signal in the 5′-UTR are shown. The gene-specific sequence illustrated is of fnuDIIM . The underlined 21 bp of longer translation signal (LTS) is from the tacp regulatory region ( 34 ) ; the short signal (STS) is a truncation of it. The LTS and downstream primers illustrated are the same as primers 5 and 6 of Table 4 ; the STS construct was made but not used in this report. Fusion of lacZ to the signal as shown creates an RBS/ATG spacing of six, within the usual range of spacing ( 35 ); lacZ native spacing is seven ( 36 ).

    Article Snippet: General materials: ∘ USERBstBI-compatible digested pMS26 (from step 1) ∘ PfuCx_TurboCx _Hotstart_DNA_polymerase (Agilent Genomics) ∘ USER enzyme (NEB M5505) ∘ PCR purification columns ∘ Universal flanking primers (glmS, ptsS ) to monitor chromosomal insertion ∘ RB ampicillin plates ∘ RB no drug plates ∘ Incubators at 30°C and 42°C ∘ SOC or other outgrowth medium Experiment-specific materials: ∘ competent host cells ∘ DNA template ∘ gene-specific primers with 5′ sequences suitable to generate USERBstBI-compatible extensions.

    Techniques: Clone Assay, Sequencing, Construct