da tailed dna  (New England Biolabs)


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    DpnI 5 000 units
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    DpnI
    DpnI 5 000 units
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    Average 89 stars, based on 17393 article reviews
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    Images

    1) Product Images from "CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants"

    Article Title: CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants

    Journal: Plant Methods

    doi: 10.1186/s13007-020-00661-x

    a In vitro CRISPR-Cas9 enrichment steps. High molecular weight nuclear DNA is extracted and crRNA probes designed. DNA 5′ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles (RNPs) bind at each side and cleave the region of interest (ROI). Double strand DNA is cleaved by Cas9 revealing blunt ends with ligatable 5′ phosphates. Cas9 remains bound to the protospacer adjacent motif (PAM)-distal end giving directionality for the strands towards the ROI. All DNA is dA-tailed, preparing the blunt ends for sequencing adapter ligation. ONT adapters are ligated to Cas9 cut sites which are both 3′ dA-tailed and 5′ phosphorylated. Library is cleaned to remove excess adapters using AMPure XP beads. Non-target molecules are not removed from the library. Library is added to the flow cell for sequencing. b Bioinformatics pipeline. Raw FAST5 reads are base called using Albacore2 (v2.3.4, ONT) and Guppy (v3.2.4, ONT) and converted to FASTQ. Reads get adapters trimmed using Porechop (v0.2.3 https://github.com/rrwick/Porechop ), corrected using Canu (v1.7) [ 36 ] and aligned to the apple reference genome (‘Golden Delicious’ double haploid GDDH13v1.1) [ 34 ] using Minimap2 (v2.9) [ 46 ] to localize physically the ‘on-target’ and ‘off-target’ enriched regions. A de novo assembly is performed by Canu (v1.7) [ 36 ] using Albacore2 (v2.3.4, ONT) corrected reads and polished using Nanopolish (v0.11.1) [ 37 ]. Canu_corrected reads and the Canu_nanopolished assembly are used as inputs to run the final assembly performed by Flye (v2.5) [ 38 ]
    Figure Legend Snippet: a In vitro CRISPR-Cas9 enrichment steps. High molecular weight nuclear DNA is extracted and crRNA probes designed. DNA 5′ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles (RNPs) bind at each side and cleave the region of interest (ROI). Double strand DNA is cleaved by Cas9 revealing blunt ends with ligatable 5′ phosphates. Cas9 remains bound to the protospacer adjacent motif (PAM)-distal end giving directionality for the strands towards the ROI. All DNA is dA-tailed, preparing the blunt ends for sequencing adapter ligation. ONT adapters are ligated to Cas9 cut sites which are both 3′ dA-tailed and 5′ phosphorylated. Library is cleaned to remove excess adapters using AMPure XP beads. Non-target molecules are not removed from the library. Library is added to the flow cell for sequencing. b Bioinformatics pipeline. Raw FAST5 reads are base called using Albacore2 (v2.3.4, ONT) and Guppy (v3.2.4, ONT) and converted to FASTQ. Reads get adapters trimmed using Porechop (v0.2.3 https://github.com/rrwick/Porechop ), corrected using Canu (v1.7) [ 36 ] and aligned to the apple reference genome (‘Golden Delicious’ double haploid GDDH13v1.1) [ 34 ] using Minimap2 (v2.9) [ 46 ] to localize physically the ‘on-target’ and ‘off-target’ enriched regions. A de novo assembly is performed by Canu (v1.7) [ 36 ] using Albacore2 (v2.3.4, ONT) corrected reads and polished using Nanopolish (v0.11.1) [ 37 ]. Canu_corrected reads and the Canu_nanopolished assembly are used as inputs to run the final assembly performed by Flye (v2.5) [ 38 ]

    Techniques Used: In Vitro, CRISPR, Molecular Weight, Ligation, Sequencing

    2) Product Images from "Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans"

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-465

    Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).
    Figure Legend Snippet: Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).

    Techniques Used: Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation, Synthesized, Staining, Activity Assay, Methylation

    3) Product Images from "Serine Catabolism by SHMT2 is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl tRNAs"

    Article Title: Serine Catabolism by SHMT2 is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl tRNAs

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.01.024

    A CRISPR/Cas9 based genetic screen identifies SHMT2 as being differentially required in low glucose conditions A, Pooled screening outline. An sgRNA library targeting 2,948 metabolic enzymes and small molecule transporters was transduced into Jurkat T cells followed by culture in Nutrostats set to 10 mM or 0.75 mM glucose for a period of 14 days. Genomic DNA was collected prior to or after the 14 day period and the abundance of sgRNAs determined by deep sequencing. B, Genes exhibiting differential essentiality in 0.75 mM glucose, compared to 10 mM glucose, median Log 2 fold change cutoff of 0.5. Genes indicated in blue are components of oxidative phosphorylation complexes, while those in green are also mitochondrially localized. C, Proliferation of Jurkat cells or clones expressing small guide RNAs targeting SHMT2 (sgSHMT2), or with reintroduction of the SHMT2 cDNA. Cells were grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). D, Immunoblot for SHMT2 or beta-actin from cell lysates of the lines described in C (above), or expressing a catalytically inactive mutant (K280A) of SHMT2 (CD SHMT2) (below). E, Data from C , normalized to the 10 mM glucose condition for each cell line. F, Proliferation as in C , using the cell lines indicated. * p
    Figure Legend Snippet: A CRISPR/Cas9 based genetic screen identifies SHMT2 as being differentially required in low glucose conditions A, Pooled screening outline. An sgRNA library targeting 2,948 metabolic enzymes and small molecule transporters was transduced into Jurkat T cells followed by culture in Nutrostats set to 10 mM or 0.75 mM glucose for a period of 14 days. Genomic DNA was collected prior to or after the 14 day period and the abundance of sgRNAs determined by deep sequencing. B, Genes exhibiting differential essentiality in 0.75 mM glucose, compared to 10 mM glucose, median Log 2 fold change cutoff of 0.5. Genes indicated in blue are components of oxidative phosphorylation complexes, while those in green are also mitochondrially localized. C, Proliferation of Jurkat cells or clones expressing small guide RNAs targeting SHMT2 (sgSHMT2), or with reintroduction of the SHMT2 cDNA. Cells were grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). D, Immunoblot for SHMT2 or beta-actin from cell lysates of the lines described in C (above), or expressing a catalytically inactive mutant (K280A) of SHMT2 (CD SHMT2) (below). E, Data from C , normalized to the 10 mM glucose condition for each cell line. F, Proliferation as in C , using the cell lines indicated. * p

    Techniques Used: CRISPR, Sequencing, Clone Assay, Expressing, Mutagenesis

    4) Product Images from "CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants"

    Article Title: CRISPR-Cas9 enrichment and long read sequencing for fine mapping in plants

    Journal: Plant Methods

    doi: 10.1186/s13007-020-00661-x

    a In vitro CRISPR-Cas9 enrichment steps. High molecular weight nuclear DNA is extracted and crRNA probes designed. DNA 5′ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles (RNPs) bind at each side and cleave the region of interest (ROI). Double strand DNA is cleaved by Cas9 revealing blunt ends with ligatable 5′ phosphates. Cas9 remains bound to the protospacer adjacent motif (PAM)-distal end giving directionality for the strands towards the ROI. All DNA is dA-tailed, preparing the blunt ends for sequencing adapter ligation. ONT adapters are ligated to Cas9 cut sites which are both 3′ dA-tailed and 5′ phosphorylated. Library is cleaned to remove excess adapters using AMPure XP beads. Non-target molecules are not removed from the library. Library is added to the flow cell for sequencing. b Bioinformatics pipeline. Raw FAST5 reads are base called using Albacore2 (v2.3.4, ONT) and Guppy (v3.2.4, ONT) and converted to FASTQ. Reads get adapters trimmed using Porechop (v0.2.3 https://github.com/rrwick/Porechop ), corrected using Canu (v1.7) [ 36 ] and aligned to the apple reference genome (‘Golden Delicious’ double haploid GDDH13v1.1) [ 34 ] using Minimap2 (v2.9) [ 46 ] to localize physically the ‘on-target’ and ‘off-target’ enriched regions. A de novo assembly is performed by Canu (v1.7) [ 36 ] using Albacore2 (v2.3.4, ONT) corrected reads and polished using Nanopolish (v0.11.1) [ 37 ]. Canu_corrected reads and the Canu_nanopolished assembly are used as inputs to run the final assembly performed by Flye (v2.5) [ 38 ]
    Figure Legend Snippet: a In vitro CRISPR-Cas9 enrichment steps. High molecular weight nuclear DNA is extracted and crRNA probes designed. DNA 5′ ends are dephosphorylated to reduce ligation of sequencing adapters to non-target strands. Cas9 ribonucleoprotein particles (RNPs) bind at each side and cleave the region of interest (ROI). Double strand DNA is cleaved by Cas9 revealing blunt ends with ligatable 5′ phosphates. Cas9 remains bound to the protospacer adjacent motif (PAM)-distal end giving directionality for the strands towards the ROI. All DNA is dA-tailed, preparing the blunt ends for sequencing adapter ligation. ONT adapters are ligated to Cas9 cut sites which are both 3′ dA-tailed and 5′ phosphorylated. Library is cleaned to remove excess adapters using AMPure XP beads. Non-target molecules are not removed from the library. Library is added to the flow cell for sequencing. b Bioinformatics pipeline. Raw FAST5 reads are base called using Albacore2 (v2.3.4, ONT) and Guppy (v3.2.4, ONT) and converted to FASTQ. Reads get adapters trimmed using Porechop (v0.2.3 https://github.com/rrwick/Porechop ), corrected using Canu (v1.7) [ 36 ] and aligned to the apple reference genome (‘Golden Delicious’ double haploid GDDH13v1.1) [ 34 ] using Minimap2 (v2.9) [ 46 ] to localize physically the ‘on-target’ and ‘off-target’ enriched regions. A de novo assembly is performed by Canu (v1.7) [ 36 ] using Albacore2 (v2.3.4, ONT) corrected reads and polished using Nanopolish (v0.11.1) [ 37 ]. Canu_corrected reads and the Canu_nanopolished assembly are used as inputs to run the final assembly performed by Flye (v2.5) [ 38 ]

    Techniques Used: In Vitro, CRISPR, Molecular Weight, Ligation, Sequencing

    5) Product Images from "Serine Catabolism by SHMT2 is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl tRNAs"

    Article Title: Serine Catabolism by SHMT2 is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl tRNAs

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.01.024

    SHMT2-null cells are unable to maintain formylmethionyl-tRNA pools in mitochondria A, Schematic overview of mitochondrial tRNA Met maturation and sensitivity to Cu 2+ or OH − treatment. Uncharged tRNA Met is charged by the mitochondrial methionyl tRNA synthetase (mtMetRS) to form Met-tRNA Met . This species is further modified by MTFMT using a formyl group (blue circle marked with an “f”), derived from serine via the SHMT2 reaction, forming fMet-tRNA Met . The fMet-tRNA Met is then used to initiate translation in the mitochondria. Upon treatment with Cu 2+ or OH − , Met-tRNA Met is hydrolyzed to tRNA Met , whereas fMet-tRNA Met is relatively resistant to Cu 2+ treatment. B–D, Detection of mitochondrial tRNA Met species by northern blot using a probe specific to the mitochondrial tRNA Met and RNA isolated from Jurkat cell clones expressing an sgRNA targeting SHMT2 or additionally re-expressing SHMT2, or clones expressing an sgRNA targeting MTHFD1L, MTHFD2, or MTHFD2L. Met and fMet modification of the tRNA alters its mobility as indicated on the left. RNA was isolated under acidic conditions to preserve fMet or Met charged species (left) or treated to selectively hydrolyze Met (Cu 2+ , middle) or non-selectively hydrolyze both fMet and Met (OH − , right).
    Figure Legend Snippet: SHMT2-null cells are unable to maintain formylmethionyl-tRNA pools in mitochondria A, Schematic overview of mitochondrial tRNA Met maturation and sensitivity to Cu 2+ or OH − treatment. Uncharged tRNA Met is charged by the mitochondrial methionyl tRNA synthetase (mtMetRS) to form Met-tRNA Met . This species is further modified by MTFMT using a formyl group (blue circle marked with an “f”), derived from serine via the SHMT2 reaction, forming fMet-tRNA Met . The fMet-tRNA Met is then used to initiate translation in the mitochondria. Upon treatment with Cu 2+ or OH − , Met-tRNA Met is hydrolyzed to tRNA Met , whereas fMet-tRNA Met is relatively resistant to Cu 2+ treatment. B–D, Detection of mitochondrial tRNA Met species by northern blot using a probe specific to the mitochondrial tRNA Met and RNA isolated from Jurkat cell clones expressing an sgRNA targeting SHMT2 or additionally re-expressing SHMT2, or clones expressing an sgRNA targeting MTHFD1L, MTHFD2, or MTHFD2L. Met and fMet modification of the tRNA alters its mobility as indicated on the left. RNA was isolated under acidic conditions to preserve fMet or Met charged species (left) or treated to selectively hydrolyze Met (Cu 2+ , middle) or non-selectively hydrolyze both fMet and Met (OH − , right).

    Techniques Used: Modification, Derivative Assay, Northern Blot, Isolation, Clone Assay, Expressing

    SHMT2 is required for proper mitochondrial respiration and translation of mitochondrially encoded proteins A, Oxygen consumption rate (OCR) of Jurkat cells cultured at the indicated glucose concentrations from a representative experiment. Jurkat cells or a clone expressing small guide RNAs targeting SHMT2 (sgSHMT2) were seeded at 200,000 cells per well immediately prior to the assay. The complex V inhibitor oligomycin (Olig), the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and the complex I and III inhibitors Antimycin (Ant) and Rotenone (Rot) were added sequentially to a final concentration of 1 μM at the indicated time points. B, Basal OCR from the lines described in A and cells re-expressing an SHMT2 cDNA. C, Proportion of OCR contributing to ATP production (measured as the decrease in OCR upon oligomycin treatment) from the lines described in A–B . D, Intracellular levels of the indicated amino acids in the indicated cell lines cultured in media containing 10 mM (high glucose) or 1.5 mM glucose (low glucose), as measured by LC/MS. E, Relative mitochondrial mass as assessed by flow cytometry based measurement of fluorescence from the indicated Jurkat cell lines stained with mitotracker green FM (75 nM). F, Relative mitochondrial membrane potential (Ψ) as assessed by flow cytometry based measurement of fluorescence from the indicated Jurkat cell lines stained with tetramethylrhodamine, methyl ester, perchlorate (200 nM). Indicated cells were treated with FCCP (20 μM). G, Relative mitochondrial DNA content in Jurkat cell lines as assessed by qPCR based measurement of the indicated mtDNA genes, normalized to an Alu repeat sequence. H, Relative mitochondrial expression in Jurkat cell lines as assessed by qPCR based measurement of the indicated mitochondrial genes. I, Immunoblot from cell lysates of the indicated lines for proteins encoded by the indicated genes. MT-CO1, MT-CO2, and MT-ND1 are encoded by the mitochondrial genome and translated in the mitochondria (MT) whereas COX4 is encoded by the nuclear genome and translated in the cytoplasm (Nuc.). J, 35 S-cysteine and 35 ) as well as subsequent immunoblotting for CO1 and CO2. * p
    Figure Legend Snippet: SHMT2 is required for proper mitochondrial respiration and translation of mitochondrially encoded proteins A, Oxygen consumption rate (OCR) of Jurkat cells cultured at the indicated glucose concentrations from a representative experiment. Jurkat cells or a clone expressing small guide RNAs targeting SHMT2 (sgSHMT2) were seeded at 200,000 cells per well immediately prior to the assay. The complex V inhibitor oligomycin (Olig), the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and the complex I and III inhibitors Antimycin (Ant) and Rotenone (Rot) were added sequentially to a final concentration of 1 μM at the indicated time points. B, Basal OCR from the lines described in A and cells re-expressing an SHMT2 cDNA. C, Proportion of OCR contributing to ATP production (measured as the decrease in OCR upon oligomycin treatment) from the lines described in A–B . D, Intracellular levels of the indicated amino acids in the indicated cell lines cultured in media containing 10 mM (high glucose) or 1.5 mM glucose (low glucose), as measured by LC/MS. E, Relative mitochondrial mass as assessed by flow cytometry based measurement of fluorescence from the indicated Jurkat cell lines stained with mitotracker green FM (75 nM). F, Relative mitochondrial membrane potential (Ψ) as assessed by flow cytometry based measurement of fluorescence from the indicated Jurkat cell lines stained with tetramethylrhodamine, methyl ester, perchlorate (200 nM). Indicated cells were treated with FCCP (20 μM). G, Relative mitochondrial DNA content in Jurkat cell lines as assessed by qPCR based measurement of the indicated mtDNA genes, normalized to an Alu repeat sequence. H, Relative mitochondrial expression in Jurkat cell lines as assessed by qPCR based measurement of the indicated mitochondrial genes. I, Immunoblot from cell lysates of the indicated lines for proteins encoded by the indicated genes. MT-CO1, MT-CO2, and MT-ND1 are encoded by the mitochondrial genome and translated in the mitochondria (MT) whereas COX4 is encoded by the nuclear genome and translated in the cytoplasm (Nuc.). J, 35 S-cysteine and 35 ) as well as subsequent immunoblotting for CO1 and CO2. * p

    Techniques Used: Cell Culture, Expressing, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Flow Cytometry, Cytometry, Fluorescence, Staining, Real-time Polymerase Chain Reaction, Sequencing

    Depletion of mitochondrial one-carbon units upon SHMT2 loss prevents proper mitochondrial translation A, Overview schematic of one-carbon metabolism indicating cytoplasmic and mitochondrial compartmentalization in wildtype cells (left), and proposed alterations upon SHMT2 deletion (right). While reactions are reversible, arrow direction and thickness indicate hypothetical relative flux upon SHMT2 deletion. Enzymes and the glycine cleavage complex (GCC) are indicated in green. Key metabolites are indicated in black. THF = tetrahydrofolate. B, E, and H, Immunoblot from cell lysates of Jurkat cell lines for proteins encoded by the indicated genes. Jurkat cell clones express sgRNAs targeting SHMT1, SHMT2, MTHFD1L, MTHFD2, or MTHFD2L or a cDNA expressing SHMT1, as indicated. For the MTHFD2L immunoblot in panel H, the arrow indicates the band corresponding to MTHFD2L protein, whereas the prominent larger band is non-specific. C and F, Above, proliferation of Jurkat cells or clones expressing the indicated sgRNAs and cDNAs. Below, data from above, normalized to the 10 mM glucose condition for each cell line. Cells were grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). D, Basal oxygen consumption rate (OCR) of the cell lines from A , cultured in 10 mM glucose. G, Basal oxygen consumption rate (OCR) of the cell lines from F , cultured in 10 mM (high glucose) or 1.5 mM glucose (low glucose). I, Basal oxygen consumption rate (OCR) of the cell lines from H , cultured in 10 mM glucose. * p
    Figure Legend Snippet: Depletion of mitochondrial one-carbon units upon SHMT2 loss prevents proper mitochondrial translation A, Overview schematic of one-carbon metabolism indicating cytoplasmic and mitochondrial compartmentalization in wildtype cells (left), and proposed alterations upon SHMT2 deletion (right). While reactions are reversible, arrow direction and thickness indicate hypothetical relative flux upon SHMT2 deletion. Enzymes and the glycine cleavage complex (GCC) are indicated in green. Key metabolites are indicated in black. THF = tetrahydrofolate. B, E, and H, Immunoblot from cell lysates of Jurkat cell lines for proteins encoded by the indicated genes. Jurkat cell clones express sgRNAs targeting SHMT1, SHMT2, MTHFD1L, MTHFD2, or MTHFD2L or a cDNA expressing SHMT1, as indicated. For the MTHFD2L immunoblot in panel H, the arrow indicates the band corresponding to MTHFD2L protein, whereas the prominent larger band is non-specific. C and F, Above, proliferation of Jurkat cells or clones expressing the indicated sgRNAs and cDNAs. Below, data from above, normalized to the 10 mM glucose condition for each cell line. Cells were grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). D, Basal oxygen consumption rate (OCR) of the cell lines from A , cultured in 10 mM glucose. G, Basal oxygen consumption rate (OCR) of the cell lines from F , cultured in 10 mM (high glucose) or 1.5 mM glucose (low glucose). I, Basal oxygen consumption rate (OCR) of the cell lines from H , cultured in 10 mM glucose. * p

    Techniques Used: Clone Assay, Expressing, Cell Culture

    Restoration of one-carbon units to the mitochondria is required to rescue mitochondrial defects observed upon SHMT2 deletion A, C, E, Immunoblot from cell lysates of Jurkat cell lines for proteins encoded by the indicated genes, upon addition of the indicated concentration of sodium formate for 3 days. B, Proliferation of Jurkat cells or clones from A , treated with the indicated concentration of sodium formate for 3 days prior to measurement. Cells were then grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). D, Basal oxygen consumption rate (OCR) of the cell lines from C , cultured in 10 mM glucose and the indicated concentration of sodium formate for 3 days. F, Above, proliferation of Jurkat cells or clones expressing the indicated sgRNAs and cDNAs. Below, data from above, normalized to the 10 mM glucose condition for each cell line. Cells were grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). * p
    Figure Legend Snippet: Restoration of one-carbon units to the mitochondria is required to rescue mitochondrial defects observed upon SHMT2 deletion A, C, E, Immunoblot from cell lysates of Jurkat cell lines for proteins encoded by the indicated genes, upon addition of the indicated concentration of sodium formate for 3 days. B, Proliferation of Jurkat cells or clones from A , treated with the indicated concentration of sodium formate for 3 days prior to measurement. Cells were then grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). D, Basal oxygen consumption rate (OCR) of the cell lines from C , cultured in 10 mM glucose and the indicated concentration of sodium formate for 3 days. F, Above, proliferation of Jurkat cells or clones expressing the indicated sgRNAs and cDNAs. Below, data from above, normalized to the 10 mM glucose condition for each cell line. Cells were grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). * p

    Techniques Used: Concentration Assay, Clone Assay, Cell Culture, Expressing

    A CRISPR/Cas9 based genetic screen identifies SHMT2 as being differentially required in low glucose conditions A, Pooled screening outline. An sgRNA library targeting 2,948 metabolic enzymes and small molecule transporters was transduced into Jurkat T cells followed by culture in Nutrostats set to 10 mM or 0.75 mM glucose for a period of 14 days. Genomic DNA was collected prior to or after the 14 day period and the abundance of sgRNAs determined by deep sequencing. B, Genes exhibiting differential essentiality in 0.75 mM glucose, compared to 10 mM glucose, median Log 2 fold change cutoff of 0.5. Genes indicated in blue are components of oxidative phosphorylation complexes, while those in green are also mitochondrially localized. C, Proliferation of Jurkat cells or clones expressing small guide RNAs targeting SHMT2 (sgSHMT2), or with reintroduction of the SHMT2 cDNA. Cells were grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). D, Immunoblot for SHMT2 or beta-actin from cell lysates of the lines described in C (above), or expressing a catalytically inactive mutant (K280A) of SHMT2 (CD SHMT2) (below). E, Data from C , normalized to the 10 mM glucose condition for each cell line. F, Proliferation as in C , using the cell lines indicated. * p
    Figure Legend Snippet: A CRISPR/Cas9 based genetic screen identifies SHMT2 as being differentially required in low glucose conditions A, Pooled screening outline. An sgRNA library targeting 2,948 metabolic enzymes and small molecule transporters was transduced into Jurkat T cells followed by culture in Nutrostats set to 10 mM or 0.75 mM glucose for a period of 14 days. Genomic DNA was collected prior to or after the 14 day period and the abundance of sgRNAs determined by deep sequencing. B, Genes exhibiting differential essentiality in 0.75 mM glucose, compared to 10 mM glucose, median Log 2 fold change cutoff of 0.5. Genes indicated in blue are components of oxidative phosphorylation complexes, while those in green are also mitochondrially localized. C, Proliferation of Jurkat cells or clones expressing small guide RNAs targeting SHMT2 (sgSHMT2), or with reintroduction of the SHMT2 cDNA. Cells were grown for 5 days in media initially containing 10 mM (high glucose) or 1.5 mM glucose (low glucose). D, Immunoblot for SHMT2 or beta-actin from cell lysates of the lines described in C (above), or expressing a catalytically inactive mutant (K280A) of SHMT2 (CD SHMT2) (below). E, Data from C , normalized to the 10 mM glucose condition for each cell line. F, Proliferation as in C , using the cell lines indicated. * p

    Techniques Used: CRISPR, Sequencing, Clone Assay, Expressing, Mutagenesis

    6) Product Images from "Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation"

    Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170724

    Deletion of an ATF3 binding region 2 Mb upstream of the IL6 gene results in specific induction of IL6 expression . (A) Overview of the construction of a dual-guide RNA (gRNA)–Cas9 expression vector. A two-step PCR amplification followed by insertion into the lentiCRISPR_v2 vector backbone was used to create a dual-expression cassette with mouse (mU6) and human (hU6) U6 promoters driving expression of two gRNAs targeting the ATF3 binding region upstream of the Il6 locus. For details, see Materials and methods. (B, top) Schematic representation of ATF3 binding region 2.07 Mb upstream of the Il6 locus, with the position of the dual gRNAs (gRNA 1 and 2) chosen for CRISPR/Cas9–mediated deletion and of the two primers (P1 and P2) used for genomic PCR analysis. (B, bottom) Nucleotide sequence of the genomic locus targeted by the two gRNAs (blue and red, respectively) together with PAM motifs (bold lines); below are the nucleotide-sequencing results of 300-bp genomic PCR products derived from two HDF clones expected to harbor the deletion (as shown in the panel below). (C) Small clusters/colonies of HDFs infected with the dual gRNAs lentiCRISPR vector (▵) and empty vector control (C) were analyzed by genomic PCR analysis with the P1 and P2 primers indicated above. Clones harboring the deletion in either a heterozygous or homozygous state, on the basis of PCR products of 300 bp versus 500 bp in size, were further analyzed by RT-qPCR for levels of Il6 , Tnc , and Tead4 expression. (D) Two additional deletion-harboring clones were analyzed together with controls by IF with Ab against IL6 and TNC. Shown are representative images together with a quantification of the fluorescence intensity signal in individual cells of deletion-harboring clones (▵) versus controls (C). n = 20 cells per condition, mean ± SEM, two-tailed unpaired t test, *, P
    Figure Legend Snippet: Deletion of an ATF3 binding region 2 Mb upstream of the IL6 gene results in specific induction of IL6 expression . (A) Overview of the construction of a dual-guide RNA (gRNA)–Cas9 expression vector. A two-step PCR amplification followed by insertion into the lentiCRISPR_v2 vector backbone was used to create a dual-expression cassette with mouse (mU6) and human (hU6) U6 promoters driving expression of two gRNAs targeting the ATF3 binding region upstream of the Il6 locus. For details, see Materials and methods. (B, top) Schematic representation of ATF3 binding region 2.07 Mb upstream of the Il6 locus, with the position of the dual gRNAs (gRNA 1 and 2) chosen for CRISPR/Cas9–mediated deletion and of the two primers (P1 and P2) used for genomic PCR analysis. (B, bottom) Nucleotide sequence of the genomic locus targeted by the two gRNAs (blue and red, respectively) together with PAM motifs (bold lines); below are the nucleotide-sequencing results of 300-bp genomic PCR products derived from two HDF clones expected to harbor the deletion (as shown in the panel below). (C) Small clusters/colonies of HDFs infected with the dual gRNAs lentiCRISPR vector (▵) and empty vector control (C) were analyzed by genomic PCR analysis with the P1 and P2 primers indicated above. Clones harboring the deletion in either a heterozygous or homozygous state, on the basis of PCR products of 300 bp versus 500 bp in size, were further analyzed by RT-qPCR for levels of Il6 , Tnc , and Tead4 expression. (D) Two additional deletion-harboring clones were analyzed together with controls by IF with Ab against IL6 and TNC. Shown are representative images together with a quantification of the fluorescence intensity signal in individual cells of deletion-harboring clones (▵) versus controls (C). n = 20 cells per condition, mean ± SEM, two-tailed unpaired t test, *, P

    Techniques Used: Binding Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, CRISPR, Sequencing, Derivative Assay, Clone Assay, Infection, Quantitative RT-PCR, Fluorescence, Two Tailed Test

    7) Product Images from "Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing"

    Article Title: Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-72

    Size fractionation of digested DNA by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with NlaIII . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.
    Figure Legend Snippet: Size fractionation of digested DNA by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with NlaIII . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.

    Techniques Used: Fractionation, In Silico, Magnetic Beads

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