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    HindIII HF
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    HindIII HF 50 000 units
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    r3104l
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    New England Biolabs hindiii hf
    HindIII HF
    HindIII HF 50 000 units
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    Average 99 stars, based on 1615 article reviews
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    Images

    1) Product Images from "Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus"

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus

    Journal: bioRxiv

    doi: 10.1101/2020.05.20.105924

    ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.
    Figure Legend Snippet: ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.

    Techniques Used: Polymerase Chain Reaction, Produced, Southern Blot, Sequencing, Plasmid Preparation

    2) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    3) Product Images from "Human herpesvirus–encoded kinase induces B cell lymphomas in vivo"

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI97053

    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.
    Figure Legend Snippet: Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Techniques Used: Transgenic Assay, Mouse Assay, Clone Assay, Plasmid Preparation, Construct, Southern Blot, Western Blot, Isolation, Polymerase Chain Reaction, Expressing, SDS Page, Positive Control

    4) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    5) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    6) Product Images from "Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells"

    Article Title: Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1445-x

    Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using symmetric ssODN donor templates and perfectly matched spacers. a Intended DNA changes at the A3 (in ALK), A11 (in EGFR), B8 (in EGFR), and B18 (in STAG2) target sites. Each red vertical line indicates the cleavage site of Cas9 nucleases, which occurs 3 bp upstream of their PAM. Each blue vertical line indicates the cleavage site of Cpf1 nucleases on one DNA strand, which occurs 18 nt downstream of their PAM. The HindIII restriction site is indicated in green . b Extent of correctly incorporating the HindIII recognition sequence into the A3, A11, or B18 target locus. Donor ssODNs with 27-nt homology arm lengths were used. The donor templates were complementary to the target DNA strand. Cells were harvested for deep sequencing analysis 72 h post-transfection. Both the Cpf1 endonucleases consistently exhibited higher levels of precise gene targeting than SpCas9. Data represent mean ± standard error of the mean (s.e.m.; n ≥ 5). *** P
    Figure Legend Snippet: Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using symmetric ssODN donor templates and perfectly matched spacers. a Intended DNA changes at the A3 (in ALK), A11 (in EGFR), B8 (in EGFR), and B18 (in STAG2) target sites. Each red vertical line indicates the cleavage site of Cas9 nucleases, which occurs 3 bp upstream of their PAM. Each blue vertical line indicates the cleavage site of Cpf1 nucleases on one DNA strand, which occurs 18 nt downstream of their PAM. The HindIII restriction site is indicated in green . b Extent of correctly incorporating the HindIII recognition sequence into the A3, A11, or B18 target locus. Donor ssODNs with 27-nt homology arm lengths were used. The donor templates were complementary to the target DNA strand. Cells were harvested for deep sequencing analysis 72 h post-transfection. Both the Cpf1 endonucleases consistently exhibited higher levels of precise gene targeting than SpCas9. Data represent mean ± standard error of the mean (s.e.m.; n ≥ 5). *** P

    Techniques Used: CRISPR, Sequencing, Transfection

    7) Product Images from "Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex"

    Article Title: Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex

    Journal: Scientific Reports

    doi: 10.1038/srep13154

    Multiple Ct TopoI can be covalently linked to the dsMCs in the presence of camptothecin. ( a ) The relaxation of a mixture of the topoisomers T −4 and T −5 by the Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) was performed in the absence (lane 2) or in the presence of 10 μM of camptothecin (CPT, lanes 3 and 4). After 10 minutes of incubation at 37 °C, SDS was added to the reaction mixtures to irreversibly trap the Ct TopoI covalently bound to DNA, and the samples were next treated with proteinase K. In lane 4, NaCl was added to the sample prior to SDS and proteinase K treatments. Samples were precipitated and analyzed on a 12% sequencing gel. dsMCs were digested with BamHI and HindIII (lane 1) or with BamHI and BglII (lane 5) and sequenced to determine the positions of the guanines and adenines in the sequence 36 . The size of single-stranded DNA fragments is indicated on the right side of the gel. lin., linear. circ., circular; ( b ) As in ( a ) except for the concentrations of camptothecin (CPT; 0 (lanes 3 and 4); 0.05 (lanes 5 and 6); 0.1 (lanes 7 and 8); 0.25 (lanes 9 and 10); 0.5 (lanes 11 and 12); 1 (lanes 13 and 14); 5 (lanes 15 and 16); 10 μM (lanes 17 and 18)) and the gel analysis (1% agarose gel, native conditions). When indicated, the samples were next treated with proteinase K (Prot K). Protein-free DNA and covalent Ct TopoI-DNA complexes are indicated. Lanes 1 and 2 contained 4% DMSO (no Ct TopoI no camptothecin); ( c ) As in ( a ) except for the concentrations of camptothecin (CPT; 0 (lane 3); 0.1 (lane 4); 0.25 (lane 5); 0.5 (lane 6); 1 (lane 7); 10 μM (lane 8)). dsMCs were digested with BamHI and HindIII (lane 1) or BamHI and BglII (lane 9) and sequenced to determine the positions of the guanines and adenines in the sequence 36 . Lane 2, mixture of T −4 and T −5 . The size of single-stranded DNA fragments is indicated on the right side of the gel.
    Figure Legend Snippet: Multiple Ct TopoI can be covalently linked to the dsMCs in the presence of camptothecin. ( a ) The relaxation of a mixture of the topoisomers T −4 and T −5 by the Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) was performed in the absence (lane 2) or in the presence of 10 μM of camptothecin (CPT, lanes 3 and 4). After 10 minutes of incubation at 37 °C, SDS was added to the reaction mixtures to irreversibly trap the Ct TopoI covalently bound to DNA, and the samples were next treated with proteinase K. In lane 4, NaCl was added to the sample prior to SDS and proteinase K treatments. Samples were precipitated and analyzed on a 12% sequencing gel. dsMCs were digested with BamHI and HindIII (lane 1) or with BamHI and BglII (lane 5) and sequenced to determine the positions of the guanines and adenines in the sequence 36 . The size of single-stranded DNA fragments is indicated on the right side of the gel. lin., linear. circ., circular; ( b ) As in ( a ) except for the concentrations of camptothecin (CPT; 0 (lanes 3 and 4); 0.05 (lanes 5 and 6); 0.1 (lanes 7 and 8); 0.25 (lanes 9 and 10); 0.5 (lanes 11 and 12); 1 (lanes 13 and 14); 5 (lanes 15 and 16); 10 μM (lanes 17 and 18)) and the gel analysis (1% agarose gel, native conditions). When indicated, the samples were next treated with proteinase K (Prot K). Protein-free DNA and covalent Ct TopoI-DNA complexes are indicated. Lanes 1 and 2 contained 4% DMSO (no Ct TopoI no camptothecin); ( c ) As in ( a ) except for the concentrations of camptothecin (CPT; 0 (lane 3); 0.1 (lane 4); 0.25 (lane 5); 0.5 (lane 6); 1 (lane 7); 10 μM (lane 8)). dsMCs were digested with BamHI and HindIII (lane 1) or BamHI and BglII (lane 9) and sequenced to determine the positions of the guanines and adenines in the sequence 36 . Lane 2, mixture of T −4 and T −5 . The size of single-stranded DNA fragments is indicated on the right side of the gel.

    Techniques Used: Cycling Probe Technology, Incubation, Sequencing, Agarose Gel Electrophoresis

    Determination of the sequence of the camptothecin-induced cleavage sites. ( a ) Identification of the camptothecin-induced cleavage sites on the BamHI-digested dsMCs. The dsMC linearized by BamHI was treated with Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) in the presence of 10 μM camptothecin as described in the methods section. After 10 minutes of incubation at 37 °C SDS was added to irreversibly trap the Ct TopoI covalently bound to DNA. The sample was next treated with proteinase K and precipitated. Lanes 5 and 10, sample directly loaded on a sequencing gel after precipitation. Sample further digested with BglII (lanes 6 and 9) or HindIII (lanes 7 and 8). dsMCs were digested with BamHI and BglII (lanes 1, 2 and 12) or BamHI and HindIII (lanes 3, 4 and 11) and sequenced to determine the positions of the guanines (lanes 1, 3 and 11) or the guanines and adenines (lanes 2, 4 and 12) in the sequence 36 . Samples loaded onto a 10% (lanes 1–7) or 7% (lanes 8–12) acrylamide sequencing gel. The camptothecin-induced cleavage sites are labelled from S 1 to S 14 ; ( b ) Overlayed scans of lanes 8 and 9 from site S 1 to S 8 . Black line, scan of lane 8 (BamHI-BglII double digestion). Blue line, scan of lane 9 (BamHI-HindIII double digestion); a. u., arbitrary unit; ( c ) The linearized dsMCs were incubated with Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) and increasing concentrations of camptothecin [CPT; 0.05 (lanes 2 and 11); 0.1 (lanes 3 and 12); 0.2 (lanes 4 and 13); 0.5 (lanes 5 and 14); 1 (lanes 6 and 15); 5 (lanes 7 and 16); 10 μM (lanes 8 and 17)] as described in the methods section. After 10 minutes of incubation at 37 °C, SDS was added to the reaction mixtures to irreversibly trap the Ct TopoI covalently bound to DNA. When indicated, the samples were next treated with proteinase K (Prot K). The products of the reactions were finally analyzed on a 1% agarose gel under native conditions. Lane 9 contained 4% DMSO (no Ct TopoI no camptothecin). Lanes 1 and 10 contained 4% DMSO instead of camptothecin.
    Figure Legend Snippet: Determination of the sequence of the camptothecin-induced cleavage sites. ( a ) Identification of the camptothecin-induced cleavage sites on the BamHI-digested dsMCs. The dsMC linearized by BamHI was treated with Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) in the presence of 10 μM camptothecin as described in the methods section. After 10 minutes of incubation at 37 °C SDS was added to irreversibly trap the Ct TopoI covalently bound to DNA. The sample was next treated with proteinase K and precipitated. Lanes 5 and 10, sample directly loaded on a sequencing gel after precipitation. Sample further digested with BglII (lanes 6 and 9) or HindIII (lanes 7 and 8). dsMCs were digested with BamHI and BglII (lanes 1, 2 and 12) or BamHI and HindIII (lanes 3, 4 and 11) and sequenced to determine the positions of the guanines (lanes 1, 3 and 11) or the guanines and adenines (lanes 2, 4 and 12) in the sequence 36 . Samples loaded onto a 10% (lanes 1–7) or 7% (lanes 8–12) acrylamide sequencing gel. The camptothecin-induced cleavage sites are labelled from S 1 to S 14 ; ( b ) Overlayed scans of lanes 8 and 9 from site S 1 to S 8 . Black line, scan of lane 8 (BamHI-BglII double digestion). Blue line, scan of lane 9 (BamHI-HindIII double digestion); a. u., arbitrary unit; ( c ) The linearized dsMCs were incubated with Ct TopoI (18.75 mU μL −1 (≈0.2 nM)) and increasing concentrations of camptothecin [CPT; 0.05 (lanes 2 and 11); 0.1 (lanes 3 and 12); 0.2 (lanes 4 and 13); 0.5 (lanes 5 and 14); 1 (lanes 6 and 15); 5 (lanes 7 and 16); 10 μM (lanes 8 and 17)] as described in the methods section. After 10 minutes of incubation at 37 °C, SDS was added to the reaction mixtures to irreversibly trap the Ct TopoI covalently bound to DNA. When indicated, the samples were next treated with proteinase K (Prot K). The products of the reactions were finally analyzed on a 1% agarose gel under native conditions. Lane 9 contained 4% DMSO (no Ct TopoI no camptothecin). Lanes 1 and 10 contained 4% DMSO instead of camptothecin.

    Techniques Used: Sequencing, Incubation, Cycling Probe Technology, Agarose Gel Electrophoresis

    8) Product Images from "MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs"

    Article Title: MYC activation and BCL2L11 silencing by a tumour virus through the large-scale reconfiguration of enhancer-promoter hubs

    Journal: eLife

    doi: 10.7554/eLife.18270

    CTCF and Cohesin binding in the MYC -556 super-enhancer region. ( A ) EBNA2 ChIP-sequencing reads in the GM12878 LCL and ( B ) Mutu III BL cells in the −556 region (as in Figure 2 ). ENCODE GM12878 ChIP-sequencing data for CTCF ( C ) and the cohesin subunits SMC3 ( D ) and RAD21 ( E ). ( F ) HindIII restriction enzyme sites and the location of the CTCF site primers used for 3C analysis ( Figure 2 ). Asterisks indicate the position of three CTCF consensus binding motifs. DOI: http://dx.doi.org/10.7554/eLife.18270.006
    Figure Legend Snippet: CTCF and Cohesin binding in the MYC -556 super-enhancer region. ( A ) EBNA2 ChIP-sequencing reads in the GM12878 LCL and ( B ) Mutu III BL cells in the −556 region (as in Figure 2 ). ENCODE GM12878 ChIP-sequencing data for CTCF ( C ) and the cohesin subunits SMC3 ( D ) and RAD21 ( E ). ( F ) HindIII restriction enzyme sites and the location of the CTCF site primers used for 3C analysis ( Figure 2 ). Asterisks indicate the position of three CTCF consensus binding motifs. DOI: http://dx.doi.org/10.7554/eLife.18270.006

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Sequencing

    9) Product Images from "Brassica yellows virus’ movement protein upregulates anthocyanin accumulation, leading to the development of purple leaf symptoms on Arabidopsis thaliana"

    Article Title: Brassica yellows virus’ movement protein upregulates anthocyanin accumulation, leading to the development of purple leaf symptoms on Arabidopsis thaliana

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34591-5

    Characterization of BrYV amplicon-transformed Arabidopsis. ( a ) Southern blot analysis indicating that the cDNA of BrYV was inserted into the Arabidopsis genome. The genomic DNA of transgenic Arabidopsis plants was digested with either Eco R I or Hin d III. (b) Northern blot results showing the constitutive expression of BrYV-encoded genomic RNA (gRNA) and subgenomic RNAs (sgRNAs). (c) Western blot analysis demonstrating the expression of the BrYV coat protein in lines 111 and 412.
    Figure Legend Snippet: Characterization of BrYV amplicon-transformed Arabidopsis. ( a ) Southern blot analysis indicating that the cDNA of BrYV was inserted into the Arabidopsis genome. The genomic DNA of transgenic Arabidopsis plants was digested with either Eco R I or Hin d III. (b) Northern blot results showing the constitutive expression of BrYV-encoded genomic RNA (gRNA) and subgenomic RNAs (sgRNAs). (c) Western blot analysis demonstrating the expression of the BrYV coat protein in lines 111 and 412.

    Techniques Used: Amplification, Transformation Assay, Southern Blot, Transgenic Assay, Northern Blot, Expressing, Western Blot

    10) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    High-throughput CUTseq. a Front picture of the I-DOT One MC, low-volume non-contact dispensing device (Dispendix) that was used in this study, and timeline for high-throughput CUTseq library preparation. IVT, in vitro transcription. The total workflow takes ~8 h for a single person to prepare 1–2 libraries, each containing up to 96 samples. The dispensing step can be done either manually or using a liquid handling device such as I-DOT One. b Number of usable reads (after alignment and PCR duplicates removal) per sample, in one multiplexed CUTseq library prepared from 96 replicate samples ( n ) of HeLa cells gDNA (5 ng), using I-DOT One. c Distribution of the sequencing error rates in the 96 replicates ( n ) shown in b . d Copy number profiles (1 Mb resolution, averaged at arm level for visualization) of 88 replicates shown in b that yielded at least 300 K usable reads. The remaining eight samples were not included, as the number of usable reads was insufficient to perform reliable copy number calling. e Distribution of all possible ( n ) pairwise Pearson’s correlations between the copy number profiles shown in d . f Fractions of the genome either amplified (AMP) or deleted (AMP) in the 88 replicates ( n ) shown in d . Each dot represents one sample. Error bars indicate the median and interquartile range. All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: High-throughput CUTseq. a Front picture of the I-DOT One MC, low-volume non-contact dispensing device (Dispendix) that was used in this study, and timeline for high-throughput CUTseq library preparation. IVT, in vitro transcription. The total workflow takes ~8 h for a single person to prepare 1–2 libraries, each containing up to 96 samples. The dispensing step can be done either manually or using a liquid handling device such as I-DOT One. b Number of usable reads (after alignment and PCR duplicates removal) per sample, in one multiplexed CUTseq library prepared from 96 replicate samples ( n ) of HeLa cells gDNA (5 ng), using I-DOT One. c Distribution of the sequencing error rates in the 96 replicates ( n ) shown in b . d Copy number profiles (1 Mb resolution, averaged at arm level for visualization) of 88 replicates shown in b that yielded at least 300 K usable reads. The remaining eight samples were not included, as the number of usable reads was insufficient to perform reliable copy number calling. e Distribution of all possible ( n ) pairwise Pearson’s correlations between the copy number profiles shown in d . f Fractions of the genome either amplified (AMP) or deleted (AMP) in the 88 replicates ( n ) shown in d . Each dot represents one sample. Error bars indicate the median and interquartile range. All the source data for this figure are provided as a Source Data file

    Techniques Used: High Throughput Screening Assay, In Vitro, Polymerase Chain Reaction, Sequencing, Amplification

    11) Product Images from "Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells"

    Article Title: Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1445-x

    Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using symmetric ssODN donor templates and perfectly matched spacers. a Intended DNA changes at the A3 (in ALK), A11 (in EGFR), B8 (in EGFR), and B18 (in STAG2) target sites. Each red vertical line indicates the cleavage site of Cas9 nucleases, which occurs 3 bp upstream of their PAM. Each blue vertical line indicates the cleavage site of Cpf1 nucleases on one DNA strand, which occurs 18 nt downstream of their PAM. The HindIII restriction site is indicated in green . b Extent of correctly incorporating the HindIII recognition sequence into the A3, A11, or B18 target locus. Donor ssODNs with 27-nt homology arm lengths were used. The donor templates were complementary to the target DNA strand. Cells were harvested for deep sequencing analysis 72 h post-transfection. Both the Cpf1 endonucleases consistently exhibited higher levels of precise gene targeting than SpCas9. Data represent mean ± standard error of the mean (s.e.m.; n ≥ 5). *** P
    Figure Legend Snippet: Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using symmetric ssODN donor templates and perfectly matched spacers. a Intended DNA changes at the A3 (in ALK), A11 (in EGFR), B8 (in EGFR), and B18 (in STAG2) target sites. Each red vertical line indicates the cleavage site of Cas9 nucleases, which occurs 3 bp upstream of their PAM. Each blue vertical line indicates the cleavage site of Cpf1 nucleases on one DNA strand, which occurs 18 nt downstream of their PAM. The HindIII restriction site is indicated in green . b Extent of correctly incorporating the HindIII recognition sequence into the A3, A11, or B18 target locus. Donor ssODNs with 27-nt homology arm lengths were used. The donor templates were complementary to the target DNA strand. Cells were harvested for deep sequencing analysis 72 h post-transfection. Both the Cpf1 endonucleases consistently exhibited higher levels of precise gene targeting than SpCas9. Data represent mean ± standard error of the mean (s.e.m.; n ≥ 5). *** P

    Techniques Used: CRISPR, Sequencing, Transfection

    12) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    13) Product Images from "Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus"

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus

    Journal: bioRxiv

    doi: 10.1101/2020.05.20.105924

    ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.
    Figure Legend Snippet: ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.

    Techniques Used: Polymerase Chain Reaction, Produced, Southern Blot, Sequencing, Plasmid Preparation

    14) Product Images from "AFM Imaging Reveals Multiple Conformational States of ADAMTS13"

    Article Title: AFM Imaging Reveals Multiple Conformational States of ADAMTS13

    Journal: Journal of Biological Engineering

    doi: 10.1186/s13036-018-0102-y

    AFM system successfully imaged proteins . ( a ) WT- and GOF-ADAMTS13 plasmids were double digested by restriction enzymes HindIII and XhoI, and verified in agarose gel electrophoresis. ( b ) Purified WT- and GOF-ADAMTS13 were analyzed by SDS-PAGE under reducing conditions on a 7.5% gel and Western blotting with anti-His antibody. ( c ) AFM images of VWF-A1 (30 kDa), BSA (67 kDa), anti-His tag antibody (150 kDa) and commercial ADAMTS13 (190 kDa). The horizontal scale bar is 200 nm. The vertical scale bar indicates the height. ( d ) The plot of volumes of these four proteins versus their molecular weights. The data was well fitted into a straight line. The goodness of fit is indicated by R 2 . According to the linear relationship y = 5.31817*x + 256.175, the volume of purified WT-ADAMTS13 (red square) corresponds to the molecular weight of 213 kDa. Data were presented as mean ± SD.
    Figure Legend Snippet: AFM system successfully imaged proteins . ( a ) WT- and GOF-ADAMTS13 plasmids were double digested by restriction enzymes HindIII and XhoI, and verified in agarose gel electrophoresis. ( b ) Purified WT- and GOF-ADAMTS13 were analyzed by SDS-PAGE under reducing conditions on a 7.5% gel and Western blotting with anti-His antibody. ( c ) AFM images of VWF-A1 (30 kDa), BSA (67 kDa), anti-His tag antibody (150 kDa) and commercial ADAMTS13 (190 kDa). The horizontal scale bar is 200 nm. The vertical scale bar indicates the height. ( d ) The plot of volumes of these four proteins versus their molecular weights. The data was well fitted into a straight line. The goodness of fit is indicated by R 2 . According to the linear relationship y = 5.31817*x + 256.175, the volume of purified WT-ADAMTS13 (red square) corresponds to the molecular weight of 213 kDa. Data were presented as mean ± SD.

    Techniques Used: Agarose Gel Electrophoresis, Purification, SDS Page, Western Blot, Molecular Weight

    15) Product Images from "Hypoxia-NOTCH1-SOX2 signaling is important for maintaining cancer stem cells in ovarian cancer"

    Article Title: Hypoxia-NOTCH1-SOX2 signaling is important for maintaining cancer stem cells in ovarian cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10954

    Hypoxia and NOTCH1 signaling increase SOX2 promoter activity A. SOX promoter activity in adherent A2780 cells after CoCl 2 treatment (100 μM, 48 h) is shown. Data indicate mean ± SD (n=4). *, P
    Figure Legend Snippet: Hypoxia and NOTCH1 signaling increase SOX2 promoter activity A. SOX promoter activity in adherent A2780 cells after CoCl 2 treatment (100 μM, 48 h) is shown. Data indicate mean ± SD (n=4). *, P

    Techniques Used: Activity Assay

    NOTCH1 and SOX2 are important for maintaining CSC properties in primary ovarian cancer cells A. RT-PCR results of spheres generated from primary epithelial ovarian cancer cells are shown with indicated probes. B. Confocal microscopy images of spheres generated from EOC-12 (left panels) and EOC-15 (right panels) after immunolabeling with anti-SOX2 antibody. Nuclei were stained with DAPI. Scale bar = 100 μm. C. Schematic outline of the sphere enrichment protocol for primary EOC culture (100 μm
    Figure Legend Snippet: NOTCH1 and SOX2 are important for maintaining CSC properties in primary ovarian cancer cells A. RT-PCR results of spheres generated from primary epithelial ovarian cancer cells are shown with indicated probes. B. Confocal microscopy images of spheres generated from EOC-12 (left panels) and EOC-15 (right panels) after immunolabeling with anti-SOX2 antibody. Nuclei were stained with DAPI. Scale bar = 100 μm. C. Schematic outline of the sphere enrichment protocol for primary EOC culture (100 μm

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Generated, Confocal Microscopy, Immunolabeling, Staining

    SOX2 expression is important for maintaining chemoresistance in ovarian cancer cells A. Viability of adherent cells (AD) or sphere cells (SP) of A2780 (upper panel) or SKOV3 (lower panel) ovarian cancer cells in the presence of increasing concentrations of paclitaxel was determined by MTT assay. The percentage of viable cells is shown after normalization to no treatment control. Data indicate mean ± SD (n=4). *, P
    Figure Legend Snippet: SOX2 expression is important for maintaining chemoresistance in ovarian cancer cells A. Viability of adherent cells (AD) or sphere cells (SP) of A2780 (upper panel) or SKOV3 (lower panel) ovarian cancer cells in the presence of increasing concentrations of paclitaxel was determined by MTT assay. The percentage of viable cells is shown after normalization to no treatment control. Data indicate mean ± SD (n=4). *, P

    Techniques Used: Expressing, MTT Assay

    NOTCH1 and SOX2 are important for maintaining CSC properties in ovarian cancer cells A. Numbers of spheres generated from adherent A2780 cells with or without CoCl 2 (100 μM) treatment in combination with SOX2 knockdown are shown. The numbers of spheres were counted on day 7 after treatment. Data indicate mean ± SD (n=4). *, P
    Figure Legend Snippet: NOTCH1 and SOX2 are important for maintaining CSC properties in ovarian cancer cells A. Numbers of spheres generated from adherent A2780 cells with or without CoCl 2 (100 μM) treatment in combination with SOX2 knockdown are shown. The numbers of spheres were counted on day 7 after treatment. Data indicate mean ± SD (n=4). *, P

    Techniques Used: Generated

    SOX2 expression is increased in spheres of ovarian cancer cells A. Spheres were generated from confluent culture of adherent SKOV3, PA-1, and A2780 cells (upper panels) and maintained in suspension culture (lower panels). Spheres were photographed using an inverted microscope on day 7 after individual sphere cells were seeded into low attachment 6-well plates. Scale bar = 100 μm. B. RT-PCR results of adherent (AD) and sphere cells (SP) with indicated probes are shown. C. RT-PCR results of A2780-SP and SKOV3-SP cells with or without SOX2 knockdown are shown with indicated probes. D. Representative images of spheres generated from A2780-SP cells with or without SOX2 knockdown are shown. Scale bar = 100 μm. E. Numbers of spheres generated from A2780-SP or SKOV3-SP cells with or without SOX2 knockdown are shown. Data indicate mean ± SD (n=4). *, P
    Figure Legend Snippet: SOX2 expression is increased in spheres of ovarian cancer cells A. Spheres were generated from confluent culture of adherent SKOV3, PA-1, and A2780 cells (upper panels) and maintained in suspension culture (lower panels). Spheres were photographed using an inverted microscope on day 7 after individual sphere cells were seeded into low attachment 6-well plates. Scale bar = 100 μm. B. RT-PCR results of adherent (AD) and sphere cells (SP) with indicated probes are shown. C. RT-PCR results of A2780-SP and SKOV3-SP cells with or without SOX2 knockdown are shown with indicated probes. D. Representative images of spheres generated from A2780-SP cells with or without SOX2 knockdown are shown. Scale bar = 100 μm. E. Numbers of spheres generated from A2780-SP or SKOV3-SP cells with or without SOX2 knockdown are shown. Data indicate mean ± SD (n=4). *, P

    Techniques Used: Expressing, Generated, Inverted Microscopy, Reverse Transcription Polymerase Chain Reaction

    16) Product Images from "Human herpesvirus–encoded kinase induces B cell lymphomas in vivo"

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI97053

    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.
    Figure Legend Snippet: Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Techniques Used: Transgenic Assay, Mouse Assay, Clone Assay, Plasmid Preparation, Construct, Southern Blot, Western Blot, Isolation, Polymerase Chain Reaction, Expressing, SDS Page, Positive Control

    17) Product Images from "Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology"

    Article Title: Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology

    Journal: eLife

    doi: 10.7554/eLife.52741

    Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after EcoRI genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain EM20C-3, invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.
    Figure Legend Snippet: Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after EcoRI genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain EM20C-3, invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.

    Techniques Used: Inverse PCR, Polymerase Chain Reaction, Sequencing, Amplification, Ligation, Fluorescence, Flow Cytometry

    18) Product Images from "FOXP1 functions as an oncogene in promoting cancer stem cell-like characteristics in ovarian cancer cells"

    Article Title: FOXP1 functions as an oncogene in promoting cancer stem cell-like characteristics in ovarian cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6510

    FOXP1 promotes the resistance of ovarian cancer cells to chemotherapeutic drugs A. The viability of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) was measured by MTT assay after treatment of cells with indicated concentrations of Paclitaxel (left panel) or Cisplatin (right panel). B. The viability of SKOV3 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by MTT assay after treatment of cells with indicated concentrations of Paclitaxel (left panel) or Cisplatin (right panel). Data are presented as mean ± SD. *, p
    Figure Legend Snippet: FOXP1 promotes the resistance of ovarian cancer cells to chemotherapeutic drugs A. The viability of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) was measured by MTT assay after treatment of cells with indicated concentrations of Paclitaxel (left panel) or Cisplatin (right panel). B. The viability of SKOV3 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by MTT assay after treatment of cells with indicated concentrations of Paclitaxel (left panel) or Cisplatin (right panel). Data are presented as mean ± SD. *, p

    Techniques Used: Over Expression, MTT Assay

    FOXP1 knockdown inhibits tumor growth in xenotransplantation of A2780 ovarian cancer cells A. Representative pictures of mice on day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown are shown. Arrows indicate the injection sites. B, C. Representative pictures (B) and the average weight (C) of tumors removed from mice on day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown into nude mice are shown. D. Measurements of tumor volume from day 14 to day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown into nude mice are shown. Data are presented as mean ± SD ( n = 6).
    Figure Legend Snippet: FOXP1 knockdown inhibits tumor growth in xenotransplantation of A2780 ovarian cancer cells A. Representative pictures of mice on day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown are shown. Arrows indicate the injection sites. B, C. Representative pictures (B) and the average weight (C) of tumors removed from mice on day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown into nude mice are shown. D. Measurements of tumor volume from day 14 to day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown into nude mice are shown. Data are presented as mean ± SD ( n = 6).

    Techniques Used: Mouse Assay, Injection

    FOXP1 promotes the spheroid formation of A2780 ovarian cancer cells A. Bright field images of spheroids generated from A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) are shown from day 5 to day 20 of spheroid culture (bar = 100 μm). B. The size of spheroids generated from A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured from day 5 to day 20 of spheroid culture. C. The number of spheroids generated from 1000 cells of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression is shown from day 5 to day 20 of spheroid culture. D. The number of cells per spheroid generated from A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression is shown from day 5 to day 20 of spheroid culture. Data are presented as mean ± SD. *, p
    Figure Legend Snippet: FOXP1 promotes the spheroid formation of A2780 ovarian cancer cells A. Bright field images of spheroids generated from A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) are shown from day 5 to day 20 of spheroid culture (bar = 100 μm). B. The size of spheroids generated from A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured from day 5 to day 20 of spheroid culture. C. The number of spheroids generated from 1000 cells of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression is shown from day 5 to day 20 of spheroid culture. D. The number of cells per spheroid generated from A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression is shown from day 5 to day 20 of spheroid culture. Data are presented as mean ± SD. *, p

    Techniques Used: Generated, Over Expression

    FOXP1 promotes expression of stemness-related genes and EMT-related genes RT-PCR analysis of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) was performed using probes for stemness-related genes A. or EMT-related genes B.
    Figure Legend Snippet: FOXP1 promotes expression of stemness-related genes and EMT-related genes RT-PCR analysis of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) was performed using probes for stemness-related genes A. or EMT-related genes B.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression

    FOXP1 promotes proliferation and migration of A2780 ovarian cancer cells A. Cell proliferation was measured by counting cells every day for four days after plating the same number (1×10 4 /well in 12-well culture plate) of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1). B, C. Migration of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by scratch wound healing assay. Bright field images (B) and quantification of wound gap (C) at 24 h, 48 h, and 72 h after application of scratch wound are shown. Wound gap was expressed as a percentage of initial wound gap. D, E. Migration of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by transwell migration assay. Fluorescence microscope images of the cell migration (bar = 100 μm) (D) and quantification of migrated cells (E) at 12 h are shown.
    Figure Legend Snippet: FOXP1 promotes proliferation and migration of A2780 ovarian cancer cells A. Cell proliferation was measured by counting cells every day for four days after plating the same number (1×10 4 /well in 12-well culture plate) of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1). B, C. Migration of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by scratch wound healing assay. Bright field images (B) and quantification of wound gap (C) at 24 h, 48 h, and 72 h after application of scratch wound are shown. Wound gap was expressed as a percentage of initial wound gap. D, E. Migration of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by transwell migration assay. Fluorescence microscope images of the cell migration (bar = 100 μm) (D) and quantification of migrated cells (E) at 12 h are shown.

    Techniques Used: Migration, Over Expression, Wound Healing Assay, Transwell Migration Assay, Fluorescence, Microscopy

    FOXP1 enhances the promoter activity of ABCG2, OCT4, NANOG, and SOX2 A–D. Luciferase reporter assay with ABCG2 promoter (A), OCT4 promoter (B), NANOG promoter (C), or SOX2 promoter (D) with or without FOXP1-binding site deletion (ΔFOXP1) was performed after co-transfection of FOXP1 and each reporter construct into A2780 ovarian cancer cells. E. Western blotting results of A2780 ovarian cancer cells with or without FOXP1 transfection are shown.
    Figure Legend Snippet: FOXP1 enhances the promoter activity of ABCG2, OCT4, NANOG, and SOX2 A–D. Luciferase reporter assay with ABCG2 promoter (A), OCT4 promoter (B), NANOG promoter (C), or SOX2 promoter (D) with or without FOXP1-binding site deletion (ΔFOXP1) was performed after co-transfection of FOXP1 and each reporter construct into A2780 ovarian cancer cells. E. Western blotting results of A2780 ovarian cancer cells with or without FOXP1 transfection are shown.

    Techniques Used: Activity Assay, Luciferase, Reporter Assay, Binding Assay, Cotransfection, Construct, Western Blot, Transfection

    Expression of FOXP1 increases in suspension culture of A2780 ovarian cancer cells A. Bright field images of the spheroid generated from A2780 ovarian cancer cells are shown from day 0 to day 20 of spheroid culture (magnification, ×100, bar = 100 μm). B. Expression of FOXP1 mRNA in the spheroids generated from A2780 ovarian cancer cells is shown by RT-PCR from day 0 to day 20 of spheroid culture. C. Expression of FOXP1 protein in the spheroids generated from A2780 ovarian cancer cells is shown by Western blotting from day 5 to day 20 of spheroid culture. D. Expressions of HIF-1α and FOXP1 in the spheroids generated from A2780 ovarian cancer cells are shown by immunocytochemistry from day 0 to day 20 of spheroid culture (bar = 50 μm). E. Results of Western blotting analysis after treating A2780 spheroid cells with DFO (100 μM) or cobalt chloride (100 μM) are shown. F. Results of quantitative analysis of FOXP1 expression in E. are shown.
    Figure Legend Snippet: Expression of FOXP1 increases in suspension culture of A2780 ovarian cancer cells A. Bright field images of the spheroid generated from A2780 ovarian cancer cells are shown from day 0 to day 20 of spheroid culture (magnification, ×100, bar = 100 μm). B. Expression of FOXP1 mRNA in the spheroids generated from A2780 ovarian cancer cells is shown by RT-PCR from day 0 to day 20 of spheroid culture. C. Expression of FOXP1 protein in the spheroids generated from A2780 ovarian cancer cells is shown by Western blotting from day 5 to day 20 of spheroid culture. D. Expressions of HIF-1α and FOXP1 in the spheroids generated from A2780 ovarian cancer cells are shown by immunocytochemistry from day 0 to day 20 of spheroid culture (bar = 50 μm). E. Results of Western blotting analysis after treating A2780 spheroid cells with DFO (100 μM) or cobalt chloride (100 μM) are shown. F. Results of quantitative analysis of FOXP1 expression in E. are shown.

    Techniques Used: Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunocytochemistry

    19) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq validation. a Copy number profiles (NlaIII, 100 kb resolution) determined with CUTseq and NEBNext using gDNA extracted from ten different FPPE tumors. BRCA, breast cancer. COAD, colon adenocarcinoma. GIST, gastrointestinal stromal tumor. MELA, melanoma. ρ , Pearson’s correlation between matched profiles. b Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in each of the paired CUTseq and NEBNext samples shown in a . Each dot represents one pair of replicates. Dashed line: linear regression. c Partitioning of all the SNVs called in two replicate (Rep) exome capture experiments using SKBR3 cells gDNA and either CUTseq or a commercial kit for library preparation (Agilent), in multiple different annotated genomic regions. Up- and downstream indicate 5 kb windows before the start codons and after the stop codons of protein-coding genes, respectively. d Same as in c , but for different substitution types. e Overlap between the high-confidence SNVs (at least 50× coverage) called in the two CUTseq replicates shown in c , d . f Overlap between all the high-confidence SNVs identified by CUTseq vs. Agilent, after merging matched replicates shown in c , d . In both e and f , the percentages refer to the total number of SNVs in the union of the two sets. All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq validation. a Copy number profiles (NlaIII, 100 kb resolution) determined with CUTseq and NEBNext using gDNA extracted from ten different FPPE tumors. BRCA, breast cancer. COAD, colon adenocarcinoma. GIST, gastrointestinal stromal tumor. MELA, melanoma. ρ , Pearson’s correlation between matched profiles. b Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in each of the paired CUTseq and NEBNext samples shown in a . Each dot represents one pair of replicates. Dashed line: linear regression. c Partitioning of all the SNVs called in two replicate (Rep) exome capture experiments using SKBR3 cells gDNA and either CUTseq or a commercial kit for library preparation (Agilent), in multiple different annotated genomic regions. Up- and downstream indicate 5 kb windows before the start codons and after the stop codons of protein-coding genes, respectively. d Same as in c , but for different substitution types. e Overlap between the high-confidence SNVs (at least 50× coverage) called in the two CUTseq replicates shown in c , d . f Overlap between all the high-confidence SNVs identified by CUTseq vs. Agilent, after merging matched replicates shown in c , d . In both e and f , the percentages refer to the total number of SNVs in the union of the two sets. All the source data for this figure are provided as a Source Data file

    Techniques Used: Amplification

    High-throughput CUTseq. a Front picture of the I-DOT One MC, low-volume non-contact dispensing device (Dispendix) that was used in this study, and timeline for high-throughput CUTseq library preparation. IVT, in vitro transcription. The total workflow takes ~8 h for a single person to prepare 1–2 libraries, each containing up to 96 samples. The dispensing step can be done either manually or using a liquid handling device such as I-DOT One. b Number of usable reads (after alignment and PCR duplicates removal) per sample, in one multiplexed CUTseq library prepared from 96 replicate samples ( n ) of HeLa cells gDNA (5 ng), using I-DOT One. c Distribution of the sequencing error rates in the 96 replicates ( n ) shown in b . d Copy number profiles (1 Mb resolution, averaged at arm level for visualization) of 88 replicates shown in b that yielded at least 300 K usable reads. The remaining eight samples were not included, as the number of usable reads was insufficient to perform reliable copy number calling. e Distribution of all possible ( n ) pairwise Pearson’s correlations between the copy number profiles shown in d . f Fractions of the genome either amplified (AMP) or deleted (AMP) in the 88 replicates ( n ) shown in d . Each dot represents one sample. Error bars indicate the median and interquartile range. All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: High-throughput CUTseq. a Front picture of the I-DOT One MC, low-volume non-contact dispensing device (Dispendix) that was used in this study, and timeline for high-throughput CUTseq library preparation. IVT, in vitro transcription. The total workflow takes ~8 h for a single person to prepare 1–2 libraries, each containing up to 96 samples. The dispensing step can be done either manually or using a liquid handling device such as I-DOT One. b Number of usable reads (after alignment and PCR duplicates removal) per sample, in one multiplexed CUTseq library prepared from 96 replicate samples ( n ) of HeLa cells gDNA (5 ng), using I-DOT One. c Distribution of the sequencing error rates in the 96 replicates ( n ) shown in b . d Copy number profiles (1 Mb resolution, averaged at arm level for visualization) of 88 replicates shown in b that yielded at least 300 K usable reads. The remaining eight samples were not included, as the number of usable reads was insufficient to perform reliable copy number calling. e Distribution of all possible ( n ) pairwise Pearson’s correlations between the copy number profiles shown in d . f Fractions of the genome either amplified (AMP) or deleted (AMP) in the 88 replicates ( n ) shown in d . Each dot represents one sample. Error bars indicate the median and interquartile range. All the source data for this figure are provided as a Source Data file

    Techniques Used: High Throughput Screening Assay, In Vitro, Polymerase Chain Reaction, Sequencing, Amplification

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    20) Product Images from "Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells"

    Article Title: Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1445-x

    Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using symmetric ssODN donor templates and perfectly matched spacers. a Intended DNA changes at the A3 (in ALK), A11 (in EGFR), B8 (in EGFR), and B18 (in STAG2) target sites. Each red vertical line indicates the cleavage site of Cas9 nucleases, which occurs 3 bp upstream of their PAM. Each blue vertical line indicates the cleavage site of Cpf1 nucleases on one DNA strand, which occurs 18 nt downstream of their PAM. The HindIII restriction site is indicated in green . b Extent of correctly incorporating the HindIII recognition sequence into the A3, A11, or B18 target locus. Donor ssODNs with 27-nt homology arm lengths were used. The donor templates were complementary to the target DNA strand. Cells were harvested for deep sequencing analysis 72 h post-transfection. Both the Cpf1 endonucleases consistently exhibited higher levels of precise gene targeting than SpCas9. Data represent mean ± standard error of the mean (s.e.m.; n ≥ 5). *** P
    Figure Legend Snippet: Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using symmetric ssODN donor templates and perfectly matched spacers. a Intended DNA changes at the A3 (in ALK), A11 (in EGFR), B8 (in EGFR), and B18 (in STAG2) target sites. Each red vertical line indicates the cleavage site of Cas9 nucleases, which occurs 3 bp upstream of their PAM. Each blue vertical line indicates the cleavage site of Cpf1 nucleases on one DNA strand, which occurs 18 nt downstream of their PAM. The HindIII restriction site is indicated in green . b Extent of correctly incorporating the HindIII recognition sequence into the A3, A11, or B18 target locus. Donor ssODNs with 27-nt homology arm lengths were used. The donor templates were complementary to the target DNA strand. Cells were harvested for deep sequencing analysis 72 h post-transfection. Both the Cpf1 endonucleases consistently exhibited higher levels of precise gene targeting than SpCas9. Data represent mean ± standard error of the mean (s.e.m.; n ≥ 5). *** P

    Techniques Used: CRISPR, Sequencing, Transfection

    21) Product Images from "AFM Imaging Reveals Multiple Conformational States of ADAMTS13"

    Article Title: AFM Imaging Reveals Multiple Conformational States of ADAMTS13

    Journal: Journal of Biological Engineering

    doi: 10.1186/s13036-018-0102-y

    AFM system successfully imaged proteins . ( a ) WT- and GOF-ADAMTS13 plasmids were double digested by restriction enzymes HindIII and XhoI, and verified in agarose gel electrophoresis. ( b ) Purified WT- and GOF-ADAMTS13 were analyzed by SDS-PAGE under reducing conditions on a 7.5% gel and Western blotting with anti-His antibody. ( c ) AFM images of VWF-A1 (30 kDa), BSA (67 kDa), anti-His tag antibody (150 kDa) and commercial ADAMTS13 (190 kDa). The horizontal scale bar is 200 nm. The vertical scale bar indicates the height. ( d ) The plot of volumes of these four proteins versus their molecular weights. The data was well fitted into a straight line. The goodness of fit is indicated by R 2 . According to the linear relationship y = 5.31817*x + 256.175, the volume of purified WT-ADAMTS13 (red square) corresponds to the molecular weight of 213 kDa. Data were presented as mean ± SD.
    Figure Legend Snippet: AFM system successfully imaged proteins . ( a ) WT- and GOF-ADAMTS13 plasmids were double digested by restriction enzymes HindIII and XhoI, and verified in agarose gel electrophoresis. ( b ) Purified WT- and GOF-ADAMTS13 were analyzed by SDS-PAGE under reducing conditions on a 7.5% gel and Western blotting with anti-His antibody. ( c ) AFM images of VWF-A1 (30 kDa), BSA (67 kDa), anti-His tag antibody (150 kDa) and commercial ADAMTS13 (190 kDa). The horizontal scale bar is 200 nm. The vertical scale bar indicates the height. ( d ) The plot of volumes of these four proteins versus their molecular weights. The data was well fitted into a straight line. The goodness of fit is indicated by R 2 . According to the linear relationship y = 5.31817*x + 256.175, the volume of purified WT-ADAMTS13 (red square) corresponds to the molecular weight of 213 kDa. Data were presented as mean ± SD.

    Techniques Used: Agarose Gel Electrophoresis, Purification, SDS Page, Western Blot, Molecular Weight

    22) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    23) Product Images from "An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.) An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants."

    Article Title: An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.) An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

    Journal: Plant, Cell & Environment

    doi: 10.1111/pce.12693

    (a) Southern blot (b) TAIL‐PCR analyses for T 0 plant VPZ‐23, five segregating T 1 plants, two homozygous T 2 plants and wild type control (WT). The final three lanes show 25 and 50 pg digested VPZ plasmid DNA with 10 μ g of digested WT DNA and 50 pg of VPZ plasmid without WT DNA. Arrows in panel b indicate the bands that were absent in WT and show a size shift between reaction 2 and 3 in the TAIL‐PCR and scored in Table 1 . TAIL‐PCR was performed with AD3 and T‐DNA specific primers RB3.
    Figure Legend Snippet: (a) Southern blot (b) TAIL‐PCR analyses for T 0 plant VPZ‐23, five segregating T 1 plants, two homozygous T 2 plants and wild type control (WT). The final three lanes show 25 and 50 pg digested VPZ plasmid DNA with 10 μ g of digested WT DNA and 50 pg of VPZ plasmid without WT DNA. Arrows in panel b indicate the bands that were absent in WT and show a size shift between reaction 2 and 3 in the TAIL‐PCR and scored in Table 1 . TAIL‐PCR was performed with AD3 and T‐DNA specific primers RB3.

    Techniques Used: Southern Blot, Polymerase Chain Reaction, Plasmid Preparation

    24) Product Images from "3C-PCR: A novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants"

    Article Title: 3C-PCR: A novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants

    Journal: Human genetics

    doi: 10.1007/s00439-017-1853-0

    Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin dIII restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key DNA fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)
    Figure Legend Snippet: Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin dIII restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key DNA fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)

    Techniques Used: Nested PCR, Ligation, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Amplification

    25) Product Images from "Brassica yellows virus’ movement protein upregulates anthocyanin accumulation, leading to the development of purple leaf symptoms on Arabidopsis thaliana"

    Article Title: Brassica yellows virus’ movement protein upregulates anthocyanin accumulation, leading to the development of purple leaf symptoms on Arabidopsis thaliana

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34591-5

    Characterization of BrYV amplicon-transformed Arabidopsis. ( a ) Southern blot analysis indicating that the cDNA of BrYV was inserted into the Arabidopsis genome. The genomic DNA of transgenic Arabidopsis plants was digested with either Eco R I or Hin d III. (b) Northern blot results showing the constitutive expression of BrYV-encoded genomic RNA (gRNA) and subgenomic RNAs (sgRNAs). (c) Western blot analysis demonstrating the expression of the BrYV coat protein in lines 111 and 412.
    Figure Legend Snippet: Characterization of BrYV amplicon-transformed Arabidopsis. ( a ) Southern blot analysis indicating that the cDNA of BrYV was inserted into the Arabidopsis genome. The genomic DNA of transgenic Arabidopsis plants was digested with either Eco R I or Hin d III. (b) Northern blot results showing the constitutive expression of BrYV-encoded genomic RNA (gRNA) and subgenomic RNAs (sgRNAs). (c) Western blot analysis demonstrating the expression of the BrYV coat protein in lines 111 and 412.

    Techniques Used: Amplification, Transformation Assay, Southern Blot, Transgenic Assay, Northern Blot, Expressing, Western Blot

    26) Product Images from "A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast"

    Article Title: A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.988105

    Guide RNA cloning strategy. The guide RNA expression vector is linearized by digestion with BamHI and HindIII. The guide RNA oligonucleotide pool is amplified by PCR with NM636 and NM637 primers to create a substrate for Gibson assembly. The assembly reaction reconstitutes an intact P(RPR1)-sgRNA expression cassette. PCR using NI-1038 will prime in the P(RPR1) region and amplify a fragment that includes the variable guide RNA sequence flanked by a portion of one Illumina sequencing adapter, corresponding to the P7 side of the library with the Read #2 primer site.
    Figure Legend Snippet: Guide RNA cloning strategy. The guide RNA expression vector is linearized by digestion with BamHI and HindIII. The guide RNA oligonucleotide pool is amplified by PCR with NM636 and NM637 primers to create a substrate for Gibson assembly. The assembly reaction reconstitutes an intact P(RPR1)-sgRNA expression cassette. PCR using NI-1038 will prime in the P(RPR1) region and amplify a fragment that includes the variable guide RNA sequence flanked by a portion of one Illumina sequencing adapter, corresponding to the P7 side of the library with the Read #2 primer site.

    Techniques Used: Clone Assay, RNA Expression, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Expressing, Sequencing

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells
    Article Snippet: .. For the RFLP analysis, 200 ng PCR products were digested overnight with either XbaI or HindIII-HF (New England Biolabs) in CutSmart buffer. .. For the RFLP analysis, 200 ng PCR products were digested overnight with either XbaI or HindIII-HF (New England Biolabs) in CutSmart buffer.

    Nucleic Acid Electrophoresis:

    Article Title: Brassica yellows virus’ movement protein upregulates anthocyanin accumulation, leading to the development of purple leaf symptoms on Arabidopsis thaliana
    Article Snippet: .. Total genomic DNA (5 μg) of Arabidopsis seedlings was digested with Eco R I-HF or Hin d III-HF (New England Biolabs) overnight at 37 °C, fragments were separated by gel electrophoresis, transferred to N+ membrane (Amersham Biosciences, Roosendaal, The Netherlands) using the capillary transfer method, hybridized with a radioactive isotopes [α-32P] dCTP-labeled cDNA probe specific for nt 5,161 to 5,620 of the 3′ BrYV fragment, recorded by phosphor autoradiography, and finally scanned using a Typhoon 9000 (GE Healthcare). .. Northern blot analysis For the detection of BrYV RNAs generated by the BrYV amplicon-transformed Arabidopsis lines, 2 μg total RNA of Col-0, line 111, and line 412 were prepared and fractionated by electrophoresis with ~5 V/cm force in a denaturing agarose gel containing formaldehyde.

    Incubation:

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus
    Article Snippet: .. Southern blotting 5 µg undigested CRF-CA D citri DNA or 5 µg CRF-CA D. citri DNA digested overnight with PstI-HF and HindIII-HF (New England Biolabs, Ibswich, Massachusetts) was electrophoresed in a 0.8% agarose/0.5x TAE gel and the gel was prepared for transfer by incubation in 0.25 M HCl for 30 minutes, then 0.5 M NaCl, 0.5 M NaOH for 30 minutes, and then 1.5 M NaCl, 0.5 M Tris-HCl pH 7 for 30 minutes. .. DNA was then transferred to an Amersham Hybond Nx membrane (GE Life Sciences, Boston, Massachusetts) for 24 hours in 20X SSC using the capillary method.

    Autoradiography:

    Article Title: Brassica yellows virus’ movement protein upregulates anthocyanin accumulation, leading to the development of purple leaf symptoms on Arabidopsis thaliana
    Article Snippet: .. Total genomic DNA (5 μg) of Arabidopsis seedlings was digested with Eco R I-HF or Hin d III-HF (New England Biolabs) overnight at 37 °C, fragments were separated by gel electrophoresis, transferred to N+ membrane (Amersham Biosciences, Roosendaal, The Netherlands) using the capillary transfer method, hybridized with a radioactive isotopes [α-32P] dCTP-labeled cDNA probe specific for nt 5,161 to 5,620 of the 3′ BrYV fragment, recorded by phosphor autoradiography, and finally scanned using a Typhoon 9000 (GE Healthcare). .. Northern blot analysis For the detection of BrYV RNAs generated by the BrYV amplicon-transformed Arabidopsis lines, 2 μg total RNA of Col-0, line 111, and line 412 were prepared and fractionated by electrophoresis with ~5 V/cm force in a denaturing agarose gel containing formaldehyde.

    Southern Blot:

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus
    Article Snippet: .. Southern blotting 5 µg undigested CRF-CA D citri DNA or 5 µg CRF-CA D. citri DNA digested overnight with PstI-HF and HindIII-HF (New England Biolabs, Ibswich, Massachusetts) was electrophoresed in a 0.8% agarose/0.5x TAE gel and the gel was prepared for transfer by incubation in 0.25 M HCl for 30 minutes, then 0.5 M NaCl, 0.5 M NaOH for 30 minutes, and then 1.5 M NaCl, 0.5 M Tris-HCl pH 7 for 30 minutes. .. DNA was then transferred to an Amersham Hybond Nx membrane (GE Life Sciences, Boston, Massachusetts) for 24 hours in 20X SSC using the capillary method.

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    New England Biolabs hindiii hf
    ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and <t>HindIII.</t> Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.
    Hindiii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.

    Journal: bioRxiv

    Article Title: Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus

    doi: 10.1101/2020.05.20.105924

    Figure Lengend Snippet: ENS is a transcribed EVE that is unevenly distributed among distinct D. citri populations. (A) Upper: PCR products produced using primers flanking ENS (primers 2 and 8). Lower: PCR products produced using primers specific to D. citri actin (primers 9 and 10). (B) Southern blot of D. citri genomic DNA using an RNA probe based on the sequence of ENS; U = undigested, D = digested with PstI and HindIII. Upper arrow denotes ENS in undigested genomic DNA. Lower arrows denote cleavage products. (C) PCR products produced using primers 3 and 7 and the indicated DNA samples. “Plasmid” is a plasmid containing the full ENS sequence and “Plasmid HindIII + SbfI” is the same plasmid digested with HindIII and SbfI. DNA was left intact or digested with an exonuclease (Exo.) prior to PCR. (D) Primers 3 or 7 were used to generate cDNA from antisense or sense transcripts, respectively. cDNAs were used as templates for PCR using primers 3 and 7. DNA or cDNA prepared without reverse transcriptase (RT) served as controls.

    Article Snippet: Southern blotting 5 µg undigested CRF-CA D citri DNA or 5 µg CRF-CA D. citri DNA digested overnight with PstI-HF and HindIII-HF (New England Biolabs, Ibswich, Massachusetts) was electrophoresed in a 0.8% agarose/0.5x TAE gel and the gel was prepared for transfer by incubation in 0.25 M HCl for 30 minutes, then 0.5 M NaCl, 0.5 M NaOH for 30 minutes, and then 1.5 M NaCl, 0.5 M Tris-HCl pH 7 for 30 minutes.

    Techniques: Polymerase Chain Reaction, Produced, Southern Blot, Sequencing, Plasmid Preparation

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Journal: Nature Communications

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    doi: 10.1038/s41467-019-12570-2

    Figure Lengend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Article Snippet: We then used I-DOT One to dispense first 5 ng diluted in 350 nl of gDNA extracted from HeLa cells, followed by 100 nl of 20 U/μl of HindIII (NEB, catalog number R3104) and 50 nl of CutSmart buffer (NEB, catalog number R3104), in 96 of the 384 wells.

    Techniques: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Journal: The Journal of Clinical Investigation

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo

    doi: 10.1172/JCI97053

    Figure Lengend Snippet: Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Article Snippet: Fifteen micrograms of DNA from each mouse was double digested with BamHI-HF and HindIII-HF (New England BioLabs) and single digested using AflII (New England BioLabs) in a total volume of 100 μl at 37°C overnight.

    Techniques: Transgenic Assay, Mouse Assay, Clone Assay, Plasmid Preparation, Construct, Southern Blot, Western Blot, Isolation, Polymerase Chain Reaction, Expressing, SDS Page, Positive Control