dnase i buffer  (New England Biolabs)


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    New England Biolabs dnase i buffer
    Identification of the VanR binding site at the promoter region of the vanABK operon (P vanABK ). The coding strand of P vanABK DNA (amplified from pJOE7658.1) was sequenced using oligonucleotide primer s8821 (A), while the noncoding strand was sequenced using oligonucleotide primer s8753 (B) according to Sanger's dideoxy chain termination method. A, C, G, and T correspond to ddATP, ddCTP, ddGTP, and ddTTP, respectively, used in the sequencing reactions. <t>DNase</t> I digestion of the coding or noncoding strands was carried out in the presence (+) or absence (−) of purified VanR. (C) Promoter sequence of the vanABK operon, including the −35 and −10 boxes (rectangles) and the transcription start site (+1; rectangle). The VanR binding site is indicated by a boldfaced line. The probable GlxR operator sequence is underlined. The start codon of VanA is depicted by an arrow.
    Dnase I Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor"

    Article Title: Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.02431-14

    Identification of the VanR binding site at the promoter region of the vanABK operon (P vanABK ). The coding strand of P vanABK DNA (amplified from pJOE7658.1) was sequenced using oligonucleotide primer s8821 (A), while the noncoding strand was sequenced using oligonucleotide primer s8753 (B) according to Sanger's dideoxy chain termination method. A, C, G, and T correspond to ddATP, ddCTP, ddGTP, and ddTTP, respectively, used in the sequencing reactions. DNase I digestion of the coding or noncoding strands was carried out in the presence (+) or absence (−) of purified VanR. (C) Promoter sequence of the vanABK operon, including the −35 and −10 boxes (rectangles) and the transcription start site (+1; rectangle). The VanR binding site is indicated by a boldfaced line. The probable GlxR operator sequence is underlined. The start codon of VanA is depicted by an arrow.
    Figure Legend Snippet: Identification of the VanR binding site at the promoter region of the vanABK operon (P vanABK ). The coding strand of P vanABK DNA (amplified from pJOE7658.1) was sequenced using oligonucleotide primer s8821 (A), while the noncoding strand was sequenced using oligonucleotide primer s8753 (B) according to Sanger's dideoxy chain termination method. A, C, G, and T correspond to ddATP, ddCTP, ddGTP, and ddTTP, respectively, used in the sequencing reactions. DNase I digestion of the coding or noncoding strands was carried out in the presence (+) or absence (−) of purified VanR. (C) Promoter sequence of the vanABK operon, including the −35 and −10 boxes (rectangles) and the transcription start site (+1; rectangle). The VanR binding site is indicated by a boldfaced line. The probable GlxR operator sequence is underlined. The start codon of VanA is depicted by an arrow.

    Techniques Used: Binding Assay, Amplification, Sequencing, Purification

    Studying the VanR binding site at P vanABK and the properties of VanR-DNA complex. (A) Mutation of the VanR binding site at P vanABK . The VanR binding site determined by DNase I footprinting is shown by boldface lines. Transcription start sites of the P vanABK wild-type sequence and the mutants thereof are enclosed by a rectangle. The start codon for eGFP is highlighted in gray. Two inverted repeats (IR1 and IR2) inside the VanR binding site are indicated by arrows. The inducibility of P vanABK and its mutants was investigated in CGXII medium with the addition of 2 mM vanillate as the inducer. Production of eGFP was measured after 6 h of induction. (B) Binding of VanR to the Cy5-labeled P vanABK wild type and its derivatives. The 2.5 nM Cy5-labeled P vanABK DNA fragment was incubated with (+) or without (−) 49.5 nM purified VanR. Cy5-labeled DNA fragments were generated by PCR using oligonucleotides s9177 and s9071. (C) The migration pattern of the Cy5-P vanABK DNA-VanR complex at the equilibrium point of reaction was studied by gel mobility shift assay. The migration of the Cy5-labeled DNA (2.5 nM) without VanR (−) was compared to the same sample with 49.5 nM VanR (+). The amount of VanR added to the gel mobility shift reactions were 29.5 nM (pJOE8297.6) and 17.4 nM (pJOE8077.1 and pJOE8300.2) according to the determined K D values.
    Figure Legend Snippet: Studying the VanR binding site at P vanABK and the properties of VanR-DNA complex. (A) Mutation of the VanR binding site at P vanABK . The VanR binding site determined by DNase I footprinting is shown by boldface lines. Transcription start sites of the P vanABK wild-type sequence and the mutants thereof are enclosed by a rectangle. The start codon for eGFP is highlighted in gray. Two inverted repeats (IR1 and IR2) inside the VanR binding site are indicated by arrows. The inducibility of P vanABK and its mutants was investigated in CGXII medium with the addition of 2 mM vanillate as the inducer. Production of eGFP was measured after 6 h of induction. (B) Binding of VanR to the Cy5-labeled P vanABK wild type and its derivatives. The 2.5 nM Cy5-labeled P vanABK DNA fragment was incubated with (+) or without (−) 49.5 nM purified VanR. Cy5-labeled DNA fragments were generated by PCR using oligonucleotides s9177 and s9071. (C) The migration pattern of the Cy5-P vanABK DNA-VanR complex at the equilibrium point of reaction was studied by gel mobility shift assay. The migration of the Cy5-labeled DNA (2.5 nM) without VanR (−) was compared to the same sample with 49.5 nM VanR (+). The amount of VanR added to the gel mobility shift reactions were 29.5 nM (pJOE8297.6) and 17.4 nM (pJOE8077.1 and pJOE8300.2) according to the determined K D values.

    Techniques Used: Binding Assay, Mutagenesis, Footprinting, Sequencing, Labeling, Incubation, Purification, Generated, Polymerase Chain Reaction, Migration, Mobility Shift

    2) Product Images from "Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing"

    Article Title: Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

    Journal: bioRxiv

    doi: 10.1101/866244

    Development and preliminary optimization of PETRI-seq ( A ) Fixation without media (brief pelleting before fixation) resulted in a higher yield (n=3, p=0.012, 2-sided t-test) of rpsB cDNA than fixation with media (formaldehyde added directly to culture). qPCR was done after in situ RT with random hexamers. ( B ) 2-minute spin before RNA purification did not alter the bulk transcriptome. Transcriptomes stabilized by RNAprotect after 2-minute spin (x-axis) was compared to ones that were stabilized immediately by either RNAprotect or flash freezing (y-axis). 2,617 operons are included, and Pearson’s r is reported. ( C ) Fixation did not alter the E. coli transcriptome. Correlation is shown between RNA purified from E. coli cells after being fixed by 4% formaldehyde for 16 hours (“Fixed Bulk”) and RNA purified directly from growing cells (“Standard Bulk”). For both libraries, reverse transcription was done after purifying the RNA. 2,617 operons are included, and Pearon’s r is reported. ( D ) We did not detect a significant change in yield of rpsB cDNA when cells were resuspended in 50% ethanol as part of cell preparation (n=2, p=0.35, 2-sided t-test). qPCR was done after cell preparation and in situ RT with gene-specific RT primer (SB10) (n=2) ( E ) Lysozyme treatment significantly improved the yield of rpsB cDNA (n=3, p=0.001, 2-sided t-test). qPCR was done after cell preparation and in situ RT with random hexamers. ( F ) qPCR after DNase treatment or incubation with DNase buffer only (“No DNase”) confirmed the efficacy of in situ DNase treatment (n=8, p=0.035, 2-sided t-test). ( G ) qPCR after cell preparation and RT with gene-specific rpsB RT primer (SB10) confirmed that DNase was inactivated, as we did not detect a significant change in the yield of rpsB cDNA with or without DNase treatment (n=2, p=0.84, 2-sided t-test). ( H ) Gel of 775-bp PCR fragment after 1-hour incubation with cells prepared for in situ RT confirmed inactivation of DNase. For the lane that was not inactivated, DNase was directly added to the incubation of cells with the PCR product. ( I ) DNase treatment did not significantly alter E. coli transcriptomes. Aggregated PETRI-seq UMIs from one library treated with DNase and one library not treated with DNase were used to calculate TPM. Both libraries used cells from the same exponential E. coli culture. ( J ) After DNase treatment and prior to RT, an aliquot of cells was lysed and column- purified. RNA integrity was assessed by bioanalyzer (DNA HS), which reported high RNA integrity number (RIN) for both E. coli and S. aureus . ( K ) Microscope images after cell preparation of E. coli . ( L ) qPCR after bulk RT and ligation with a 16-base or 30-base linker confirmed that ligation was effective with a 16-base linker. We detected a mild increase in ligation efficiency with the 16-base linker (p=0.001, n=3, 2-sided t-test), though the fold-change between the conditions was minor (1.5x). ΔΔCt was calculated for ligated product relative to total RT product and normalized to cDNA prepared with an RT primer including the ligated sequence (SB114). ( M ) qPCR after cell preparation and in situ RT showed that cDNA was retained after AMPure purification (n=4, p=0.69, 2-sided t-test). ( N,O ) Number of mRNA UMIs (K) or operons (J) per BC after PETRI-seq with second strand synthesis or template switch used for library preparation. Second-strand synthesis resulted in significantly more mRNAs per cell (p
    Figure Legend Snippet: Development and preliminary optimization of PETRI-seq ( A ) Fixation without media (brief pelleting before fixation) resulted in a higher yield (n=3, p=0.012, 2-sided t-test) of rpsB cDNA than fixation with media (formaldehyde added directly to culture). qPCR was done after in situ RT with random hexamers. ( B ) 2-minute spin before RNA purification did not alter the bulk transcriptome. Transcriptomes stabilized by RNAprotect after 2-minute spin (x-axis) was compared to ones that were stabilized immediately by either RNAprotect or flash freezing (y-axis). 2,617 operons are included, and Pearson’s r is reported. ( C ) Fixation did not alter the E. coli transcriptome. Correlation is shown between RNA purified from E. coli cells after being fixed by 4% formaldehyde for 16 hours (“Fixed Bulk”) and RNA purified directly from growing cells (“Standard Bulk”). For both libraries, reverse transcription was done after purifying the RNA. 2,617 operons are included, and Pearon’s r is reported. ( D ) We did not detect a significant change in yield of rpsB cDNA when cells were resuspended in 50% ethanol as part of cell preparation (n=2, p=0.35, 2-sided t-test). qPCR was done after cell preparation and in situ RT with gene-specific RT primer (SB10) (n=2) ( E ) Lysozyme treatment significantly improved the yield of rpsB cDNA (n=3, p=0.001, 2-sided t-test). qPCR was done after cell preparation and in situ RT with random hexamers. ( F ) qPCR after DNase treatment or incubation with DNase buffer only (“No DNase”) confirmed the efficacy of in situ DNase treatment (n=8, p=0.035, 2-sided t-test). ( G ) qPCR after cell preparation and RT with gene-specific rpsB RT primer (SB10) confirmed that DNase was inactivated, as we did not detect a significant change in the yield of rpsB cDNA with or without DNase treatment (n=2, p=0.84, 2-sided t-test). ( H ) Gel of 775-bp PCR fragment after 1-hour incubation with cells prepared for in situ RT confirmed inactivation of DNase. For the lane that was not inactivated, DNase was directly added to the incubation of cells with the PCR product. ( I ) DNase treatment did not significantly alter E. coli transcriptomes. Aggregated PETRI-seq UMIs from one library treated with DNase and one library not treated with DNase were used to calculate TPM. Both libraries used cells from the same exponential E. coli culture. ( J ) After DNase treatment and prior to RT, an aliquot of cells was lysed and column- purified. RNA integrity was assessed by bioanalyzer (DNA HS), which reported high RNA integrity number (RIN) for both E. coli and S. aureus . ( K ) Microscope images after cell preparation of E. coli . ( L ) qPCR after bulk RT and ligation with a 16-base or 30-base linker confirmed that ligation was effective with a 16-base linker. We detected a mild increase in ligation efficiency with the 16-base linker (p=0.001, n=3, 2-sided t-test), though the fold-change between the conditions was minor (1.5x). ΔΔCt was calculated for ligated product relative to total RT product and normalized to cDNA prepared with an RT primer including the ligated sequence (SB114). ( M ) qPCR after cell preparation and in situ RT showed that cDNA was retained after AMPure purification (n=4, p=0.69, 2-sided t-test). ( N,O ) Number of mRNA UMIs (K) or operons (J) per BC after PETRI-seq with second strand synthesis or template switch used for library preparation. Second-strand synthesis resulted in significantly more mRNAs per cell (p

    Techniques Used: Real-time Polymerase Chain Reaction, In Situ, Purification, Incubation, Polymerase Chain Reaction, Microscopy, Ligation, Sequencing

    3) Product Images from "The melREDCA Operon Encodes a Utilization System for the Raffinose Family of Oligosaccharides in Bacillus subtilis"

    Article Title: The melREDCA Operon Encodes a Utilization System for the Raffinose Family of Oligosaccharides in Bacillus subtilis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00109-19

    Characterization of P msmR . (A) The DNA sequence between msmR and ytaP start codons is shown. The open arrows show the start codons of ytaP and msmR . The core elements of P msmR (−35 and −10 boxes) and the transcription start site of msmR are indicated with boxes. The protected regions of P msmR DNA by MsmR from DNase I digestion (FP1 and FP2) are in red letters. The inverted repeats within the MsmR binding site are indicated by solid arrows. The putative cre site (gray highlighted) is also demonstrated. The green boxes show the DNA regions used for the electrophoretic mobility shift assay. (B) Identification of the transcription start site (TSS) of msmR was performed by primer extension. The migration of the generated cDNA fragment (orange) was compared with the sequencing reaction. (C) An electrophoretic mobility shift assay was carried out using 5′-end Cy5-labeled DNA fragments of P msmR , the FP1 inverted repeat, and the FP2 inverted repeat. The amplified DNA fragment from the GlcR binding site was used as a negative control. The migration of the DNA fragment was investigated in the absence (−) or presence (+) of MsmR. (D) The chromatographs of the DNA footprinting and DNA sequencing reactions are separately shown. The 6-FAM-labeled P msmR DNA was digested with DNase I in the absence (orange) or presence (blue) of 0.27 mM MsmR. The identified DNA footprints, FP1 and FP2, were then compared with the DNA sequencing reaction utilizing ddATP (green), ddGTP (black), ddCTP (blue), and ddTTP (red).
    Figure Legend Snippet: Characterization of P msmR . (A) The DNA sequence between msmR and ytaP start codons is shown. The open arrows show the start codons of ytaP and msmR . The core elements of P msmR (−35 and −10 boxes) and the transcription start site of msmR are indicated with boxes. The protected regions of P msmR DNA by MsmR from DNase I digestion (FP1 and FP2) are in red letters. The inverted repeats within the MsmR binding site are indicated by solid arrows. The putative cre site (gray highlighted) is also demonstrated. The green boxes show the DNA regions used for the electrophoretic mobility shift assay. (B) Identification of the transcription start site (TSS) of msmR was performed by primer extension. The migration of the generated cDNA fragment (orange) was compared with the sequencing reaction. (C) An electrophoretic mobility shift assay was carried out using 5′-end Cy5-labeled DNA fragments of P msmR , the FP1 inverted repeat, and the FP2 inverted repeat. The amplified DNA fragment from the GlcR binding site was used as a negative control. The migration of the DNA fragment was investigated in the absence (−) or presence (+) of MsmR. (D) The chromatographs of the DNA footprinting and DNA sequencing reactions are separately shown. The 6-FAM-labeled P msmR DNA was digested with DNase I in the absence (orange) or presence (blue) of 0.27 mM MsmR. The identified DNA footprints, FP1 and FP2, were then compared with the DNA sequencing reaction utilizing ddATP (green), ddGTP (black), ddCTP (blue), and ddTTP (red).

    Techniques Used: Sequencing, Binding Assay, Electrophoretic Mobility Shift Assay, Migration, Generated, Labeling, Amplification, Negative Control, DNA Footprinting, DNA Sequencing

    4) Product Images from "Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing"

    Article Title: Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

    Journal: Nature microbiology

    doi: 10.1038/s41564-020-0729-6

    Development and preliminary optimization of PETRI-seq ( a ) qPCR after in situ RT with random hexamers shows higher yield of rpsB cDNA from fixation without media (pelleting before) than fixation with media (formaldehyde added to culture) [n=3 technically independent samples (dots), p=0.012, 2-sided t-test]. Bars show mean abundance. ( b ) Transcriptome stabilized by RNAprotect after 2-minute spin was highly correlated with transcriptomes stabilized immediately by either RNAprotect or flash freezing. Pearson’s r is reported. ( c ) RNA purified from E. coli cells after 16-hour 4% formaldehyde fixation (“Fixed Bulk”) was highly correlated with non-fixed RNA (“Standard Bulk”). 2,617 operons included. Pearson’s r is reported. ( d ) qPCR after in situ RT with rpsB -specific primer (SB10) showed similar yield when cells were resuspended in 50% ethanol (n=2 technically independent samples). ( e ) qPCR after in situ RT with random hexamers shows improved yield of rpsB cDNA after lysozyme treatment (n=3 technically independent samples [dots], p=0.001, 2-sided t-test). Bars show mean abundance. ( f ) qPCR after DNase treatment or incubation with only DNase buffer confirmed in situ DNase treatment efficacy (n=8 technically independent samples [dots], p=0.035, 2-sided t-test). Bars show mean abundance. ( g ) qPCR after in situ RT with rpsB -specific primer (SB10) confirmed DNase inactivation, as yield was unchanged (n=2 technically independent samples [dots]). Bars show mean proportion. ( h ) Gel of 775-bp PCR fragment after 1-hour incubation with DNase-treated cells confirmed DNase inactivation. Right-most lane: DNase was directly added to PCR product. Experiment conducted one time. ( i ) Aggregated PETRI-seq UMIs from DNase-treated and untreated libraries were highly correlated. Pearson’s r reported. ( j ) Bioanalyzer traces of RNA purified after in situ DNase treatment and cell lysis ( methods ). ( k ) Imaging after E. coli cell preparation. Images for all libraries looked similar (n=8). ( l ) qPCR after bulk RT and ligation ( methods ) confirmed effective ligation with a 16-base linker. Minor increase (1.5×) in ligation efficiency was detected (p=0.001, n=3 technically independent samples [dots], 2-sided t-test). Bars show mean proportion. ( m ) qPCR after in situ RT showed cDNA retention after AMPure purification (n=4 technically independent samples, p=0.69, 2-sided t-test). Bars show mean abundance. ( n,o ) Second-strand synthesis yielded more mRNAs and operons per cell (p
    Figure Legend Snippet: Development and preliminary optimization of PETRI-seq ( a ) qPCR after in situ RT with random hexamers shows higher yield of rpsB cDNA from fixation without media (pelleting before) than fixation with media (formaldehyde added to culture) [n=3 technically independent samples (dots), p=0.012, 2-sided t-test]. Bars show mean abundance. ( b ) Transcriptome stabilized by RNAprotect after 2-minute spin was highly correlated with transcriptomes stabilized immediately by either RNAprotect or flash freezing. Pearson’s r is reported. ( c ) RNA purified from E. coli cells after 16-hour 4% formaldehyde fixation (“Fixed Bulk”) was highly correlated with non-fixed RNA (“Standard Bulk”). 2,617 operons included. Pearson’s r is reported. ( d ) qPCR after in situ RT with rpsB -specific primer (SB10) showed similar yield when cells were resuspended in 50% ethanol (n=2 technically independent samples). ( e ) qPCR after in situ RT with random hexamers shows improved yield of rpsB cDNA after lysozyme treatment (n=3 technically independent samples [dots], p=0.001, 2-sided t-test). Bars show mean abundance. ( f ) qPCR after DNase treatment or incubation with only DNase buffer confirmed in situ DNase treatment efficacy (n=8 technically independent samples [dots], p=0.035, 2-sided t-test). Bars show mean abundance. ( g ) qPCR after in situ RT with rpsB -specific primer (SB10) confirmed DNase inactivation, as yield was unchanged (n=2 technically independent samples [dots]). Bars show mean proportion. ( h ) Gel of 775-bp PCR fragment after 1-hour incubation with DNase-treated cells confirmed DNase inactivation. Right-most lane: DNase was directly added to PCR product. Experiment conducted one time. ( i ) Aggregated PETRI-seq UMIs from DNase-treated and untreated libraries were highly correlated. Pearson’s r reported. ( j ) Bioanalyzer traces of RNA purified after in situ DNase treatment and cell lysis ( methods ). ( k ) Imaging after E. coli cell preparation. Images for all libraries looked similar (n=8). ( l ) qPCR after bulk RT and ligation ( methods ) confirmed effective ligation with a 16-base linker. Minor increase (1.5×) in ligation efficiency was detected (p=0.001, n=3 technically independent samples [dots], 2-sided t-test). Bars show mean proportion. ( m ) qPCR after in situ RT showed cDNA retention after AMPure purification (n=4 technically independent samples, p=0.69, 2-sided t-test). Bars show mean abundance. ( n,o ) Second-strand synthesis yielded more mRNAs and operons per cell (p

    Techniques Used: Real-time Polymerase Chain Reaction, In Situ, Purification, Incubation, Polymerase Chain Reaction, Lysis, Imaging, Ligation

    5) Product Images from "Glucose-Dependent Activation of Bacillus anthracis Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ †"

    Article Title: Glucose-Dependent Activation of Bacillus anthracis Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ Toxin Gene Expression and Virulence Requires the Carbon Catabolite Protein CcpA ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01656-09

    DNase I footprinting analysis of CcpA binding to the atxA (A and B) and BAS3893 (C and D) promoters. Fragments labeled with γ- 32 P at the 5′ end (coding strand) (A and C) or at the 3′ end (noncoding strand) (B and D) were incubated
    Figure Legend Snippet: DNase I footprinting analysis of CcpA binding to the atxA (A and B) and BAS3893 (C and D) promoters. Fragments labeled with γ- 32 P at the 5′ end (coding strand) (A and C) or at the 3′ end (noncoding strand) (B and D) were incubated

    Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

    6) Product Images from "Enterococcus faecalis Virulence Regulator FsrA Binding to Target Promoters ▿"

    Article Title: Enterococcus faecalis Virulence Regulator FsrA Binding to Target Promoters ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01522-10

    DNase I footprinting analysis of the promoter-regulatory regions of fsrB (A) and gelE (B). The labeled DNA fragments were obtained as described in Materials and Methods. The fragments were incubated with different amounts of FsrA-His6 before DNase I treatment.
    Figure Legend Snippet: DNase I footprinting analysis of the promoter-regulatory regions of fsrB (A) and gelE (B). The labeled DNA fragments were obtained as described in Materials and Methods. The fragments were incubated with different amounts of FsrA-His6 before DNase I treatment.

    Techniques Used: Footprinting, Labeling, Incubation

    7) Product Images from "A peculiar IclR family transcription factor regulates para-hydroxybenzoate catabolism in Streptomyces coelicolor"

    Article Title: A peculiar IclR family transcription factor regulates para-hydroxybenzoate catabolism in Streptomyces coelicolor

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1234

    Biochemical and bioinformatic analyses of PobR, a negative regulator of pobA . ( A ) Predicted domains organization of PobR protein. Both N-terminal domain and C-terminal domains of PobR are homologous to intact IclR family transcription factors. ( B ) The overall view of the region upstream of the pobA open reading frame (ORF), highlighting the transcription start site of pobA (turquoise), putative -35 and -10 pobA promoter elements (underlined and highlighted in orange) and the PobR regulator binding site sequence (in bold and boxed). ( C ) DNA binding of PobR was assessed using agarose electrophoretic mobility shift assays (EMSAs). A PCR-amplified DNA fragment spanning the entire region between the transcription and translation start sites (5′-untranslated region) was titrated with PobR. In comparison with the unbound DNA probe in the electrophoresis, retarded migration of the DNA probe was observed upon addition of PobR. Bands representing the PobR-DNA probe complex and the unbound DNA probe are highlighted by arrows. For a negative control, a 486-bp fragment amplified from hrdB ORF, which does not contain the PobR binding site, was used. ( D ) The PobR binding site upstream of the pobA ORF was mapped using DNase I footprinting analysis. A 5′- 32 P end-labeled 118-bp DNA fragment was digested with DNase I in the absence and presence of heterologously produced and purified PobR. In comparison with the DNA digest in the absence of PobR, a protected region was observed when DNA was incubated with PobR before digestion. Sequencing ladders indicated by lanes C/T and A/G were generated via Maxam-Gilbert reactions. The reactions were performed in duplicate. ( E ) Sequence alignment of the regions upstream of the pobA ORFs in various streptomycetes. Putative –35 and –10 pobA promoter elements and PobR binding site sequence (boxed) identified in DNase I footprinting reaction are conserved in several members of the Streptomyces genus. A conserved PobR binding site was also identified using the Gibbs Motif Sampler ( 47 ). Sequence logo images ( 51 ) depict the sequence conservation of PobR binding site.
    Figure Legend Snippet: Biochemical and bioinformatic analyses of PobR, a negative regulator of pobA . ( A ) Predicted domains organization of PobR protein. Both N-terminal domain and C-terminal domains of PobR are homologous to intact IclR family transcription factors. ( B ) The overall view of the region upstream of the pobA open reading frame (ORF), highlighting the transcription start site of pobA (turquoise), putative -35 and -10 pobA promoter elements (underlined and highlighted in orange) and the PobR regulator binding site sequence (in bold and boxed). ( C ) DNA binding of PobR was assessed using agarose electrophoretic mobility shift assays (EMSAs). A PCR-amplified DNA fragment spanning the entire region between the transcription and translation start sites (5′-untranslated region) was titrated with PobR. In comparison with the unbound DNA probe in the electrophoresis, retarded migration of the DNA probe was observed upon addition of PobR. Bands representing the PobR-DNA probe complex and the unbound DNA probe are highlighted by arrows. For a negative control, a 486-bp fragment amplified from hrdB ORF, which does not contain the PobR binding site, was used. ( D ) The PobR binding site upstream of the pobA ORF was mapped using DNase I footprinting analysis. A 5′- 32 P end-labeled 118-bp DNA fragment was digested with DNase I in the absence and presence of heterologously produced and purified PobR. In comparison with the DNA digest in the absence of PobR, a protected region was observed when DNA was incubated with PobR before digestion. Sequencing ladders indicated by lanes C/T and A/G were generated via Maxam-Gilbert reactions. The reactions were performed in duplicate. ( E ) Sequence alignment of the regions upstream of the pobA ORFs in various streptomycetes. Putative –35 and –10 pobA promoter elements and PobR binding site sequence (boxed) identified in DNase I footprinting reaction are conserved in several members of the Streptomyces genus. A conserved PobR binding site was also identified using the Gibbs Motif Sampler ( 47 ). Sequence logo images ( 51 ) depict the sequence conservation of PobR binding site.

    Techniques Used: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Polymerase Chain Reaction, Amplification, Electrophoresis, Migration, Negative Control, Footprinting, Labeling, Produced, Purification, Incubation, Generated

    8) Product Images from "High-throughput molecular recording can determine the identity and biological activity of sequences within single cells"

    Article Title: High-throughput molecular recording can determine the identity and biological activity of sequences within single cells

    Journal: bioRxiv

    doi: 10.1101/2022.03.09.483646

    Validation of DHARMA performance across 24 promoters with a wide range of activities. DNA fragments containing twenty-four promoters were synthesized and cloned into a plasmid as shown in Figure 1 , such that identical promoters drove the expression of base editor and GFP. The resulting constructs were separately transformed into electrocompetent NEB 10-beta cells which already harbor a sgRNA expression plasmid. After 1 h of outgrowth, cells were treated with DNaseI and resuspended in media containing chlo-ramphenicol and kanamycin. Samples were taken approximately every hour after outgrowth for fluorescence measurement and barcoding PCR amplification of the canvas sequences. (a) Accumulation of C to T mutations in the canvas region of each construct. Canvas regions were amplified using barcoded primers and sequenced on a MinION R10.4 flow cell. Demultiplexed reads were aligned to the reference canvas sequence and numbers of C to T mismatches were counted and normalized against total number of full-length reads for each sample. 0.5-0.8 volumes of culture were replaced with fresh media at each sampling time point to maintain log-phase growth. (b) The log-transformed total number of C to T mutations in the canvas region at 4.5h after electroporation was plotted against the corresponding log-transformed GFP fluorescence intensity for each library member. Inset: plot of insulated promoters.
    Figure Legend Snippet: Validation of DHARMA performance across 24 promoters with a wide range of activities. DNA fragments containing twenty-four promoters were synthesized and cloned into a plasmid as shown in Figure 1 , such that identical promoters drove the expression of base editor and GFP. The resulting constructs were separately transformed into electrocompetent NEB 10-beta cells which already harbor a sgRNA expression plasmid. After 1 h of outgrowth, cells were treated with DNaseI and resuspended in media containing chlo-ramphenicol and kanamycin. Samples were taken approximately every hour after outgrowth for fluorescence measurement and barcoding PCR amplification of the canvas sequences. (a) Accumulation of C to T mutations in the canvas region of each construct. Canvas regions were amplified using barcoded primers and sequenced on a MinION R10.4 flow cell. Demultiplexed reads were aligned to the reference canvas sequence and numbers of C to T mismatches were counted and normalized against total number of full-length reads for each sample. 0.5-0.8 volumes of culture were replaced with fresh media at each sampling time point to maintain log-phase growth. (b) The log-transformed total number of C to T mutations in the canvas region at 4.5h after electroporation was plotted against the corresponding log-transformed GFP fluorescence intensity for each library member. Inset: plot of insulated promoters.

    Techniques Used: Synthesized, Clone Assay, Plasmid Preparation, Expressing, Construct, Transformation Assay, Fluorescence, Polymerase Chain Reaction, Amplification, Sequencing, Sampling, Electroporation

    9) Product Images from "Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing"

    Article Title: Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

    Journal: bioRxiv

    doi: 10.1101/866244

    Development and preliminary optimization of PETRI-seq ( A ) Fixation without media (brief pelleting before fixation) resulted in a higher yield (n=3, p=0.012, 2-sided t-test) of rpsB cDNA than fixation with media (formaldehyde added directly to culture). qPCR was done after in situ RT with random hexamers. ( B ) 2-minute spin before RNA purification did not alter the bulk transcriptome. Transcriptomes stabilized by RNAprotect after 2-minute spin (x-axis) was compared to ones that were stabilized immediately by either RNAprotect or flash freezing (y-axis). 2,617 operons are included, and Pearson’s r is reported. ( C ) Fixation did not alter the E. coli transcriptome. Correlation is shown between RNA purified from E. coli cells after being fixed by 4% formaldehyde for 16 hours (“Fixed Bulk”) and RNA purified directly from growing cells (“Standard Bulk”). For both libraries, reverse transcription was done after purifying the RNA. 2,617 operons are included, and Pearon’s r is reported. ( D ) We did not detect a significant change in yield of rpsB cDNA when cells were resuspended in 50% ethanol as part of cell preparation (n=2, p=0.35, 2-sided t-test). qPCR was done after cell preparation and in situ RT with gene-specific RT primer (SB10) (n=2) ( E ) Lysozyme treatment significantly improved the yield of rpsB cDNA (n=3, p=0.001, 2-sided t-test). qPCR was done after cell preparation and in situ RT with random hexamers. ( F ) qPCR after DNase treatment or incubation with DNase buffer only (“No DNase”) confirmed the efficacy of in situ DNase treatment (n=8, p=0.035, 2-sided t-test). ( G ) qPCR after cell preparation and RT with gene-specific rpsB RT primer (SB10) confirmed that DNase was inactivated, as we did not detect a significant change in the yield of rpsB cDNA with or without DNase treatment (n=2, p=0.84, 2-sided t-test). ( H ) Gel of 775-bp PCR fragment after 1-hour incubation with cells prepared for in situ RT confirmed inactivation of DNase. For the lane that was not inactivated, DNase was directly added to the incubation of cells with the PCR product. ( I ) DNase treatment did not significantly alter E. coli transcriptomes. Aggregated PETRI-seq UMIs from one library treated with DNase and one library not treated with DNase were used to calculate TPM. Both libraries used cells from the same exponential E. coli culture. ( J ) After DNase treatment and prior to RT, an aliquot of cells was lysed and column- purified. RNA integrity was assessed by bioanalyzer (DNA HS), which reported high RNA integrity number (RIN) for both E. coli and S. aureus . ( K ) Microscope images after cell preparation of E. coli . ( L ) qPCR after bulk RT and ligation with a 16-base or 30-base linker confirmed that ligation was effective with a 16-base linker. We detected a mild increase in ligation efficiency with the 16-base linker (p=0.001, n=3, 2-sided t-test), though the fold-change between the conditions was minor (1.5x). ΔΔCt was calculated for ligated product relative to total RT product and normalized to cDNA prepared with an RT primer including the ligated sequence (SB114). ( M ) qPCR after cell preparation and in situ RT showed that cDNA was retained after AMPure purification (n=4, p=0.69, 2-sided t-test). ( N,O ) Number of mRNA UMIs (K) or operons (J) per BC after PETRI-seq with second strand synthesis or template switch used for library preparation. Second-strand synthesis resulted in significantly more mRNAs per cell (p
    Figure Legend Snippet: Development and preliminary optimization of PETRI-seq ( A ) Fixation without media (brief pelleting before fixation) resulted in a higher yield (n=3, p=0.012, 2-sided t-test) of rpsB cDNA than fixation with media (formaldehyde added directly to culture). qPCR was done after in situ RT with random hexamers. ( B ) 2-minute spin before RNA purification did not alter the bulk transcriptome. Transcriptomes stabilized by RNAprotect after 2-minute spin (x-axis) was compared to ones that were stabilized immediately by either RNAprotect or flash freezing (y-axis). 2,617 operons are included, and Pearson’s r is reported. ( C ) Fixation did not alter the E. coli transcriptome. Correlation is shown between RNA purified from E. coli cells after being fixed by 4% formaldehyde for 16 hours (“Fixed Bulk”) and RNA purified directly from growing cells (“Standard Bulk”). For both libraries, reverse transcription was done after purifying the RNA. 2,617 operons are included, and Pearon’s r is reported. ( D ) We did not detect a significant change in yield of rpsB cDNA when cells were resuspended in 50% ethanol as part of cell preparation (n=2, p=0.35, 2-sided t-test). qPCR was done after cell preparation and in situ RT with gene-specific RT primer (SB10) (n=2) ( E ) Lysozyme treatment significantly improved the yield of rpsB cDNA (n=3, p=0.001, 2-sided t-test). qPCR was done after cell preparation and in situ RT with random hexamers. ( F ) qPCR after DNase treatment or incubation with DNase buffer only (“No DNase”) confirmed the efficacy of in situ DNase treatment (n=8, p=0.035, 2-sided t-test). ( G ) qPCR after cell preparation and RT with gene-specific rpsB RT primer (SB10) confirmed that DNase was inactivated, as we did not detect a significant change in the yield of rpsB cDNA with or without DNase treatment (n=2, p=0.84, 2-sided t-test). ( H ) Gel of 775-bp PCR fragment after 1-hour incubation with cells prepared for in situ RT confirmed inactivation of DNase. For the lane that was not inactivated, DNase was directly added to the incubation of cells with the PCR product. ( I ) DNase treatment did not significantly alter E. coli transcriptomes. Aggregated PETRI-seq UMIs from one library treated with DNase and one library not treated with DNase were used to calculate TPM. Both libraries used cells from the same exponential E. coli culture. ( J ) After DNase treatment and prior to RT, an aliquot of cells was lysed and column- purified. RNA integrity was assessed by bioanalyzer (DNA HS), which reported high RNA integrity number (RIN) for both E. coli and S. aureus . ( K ) Microscope images after cell preparation of E. coli . ( L ) qPCR after bulk RT and ligation with a 16-base or 30-base linker confirmed that ligation was effective with a 16-base linker. We detected a mild increase in ligation efficiency with the 16-base linker (p=0.001, n=3, 2-sided t-test), though the fold-change between the conditions was minor (1.5x). ΔΔCt was calculated for ligated product relative to total RT product and normalized to cDNA prepared with an RT primer including the ligated sequence (SB114). ( M ) qPCR after cell preparation and in situ RT showed that cDNA was retained after AMPure purification (n=4, p=0.69, 2-sided t-test). ( N,O ) Number of mRNA UMIs (K) or operons (J) per BC after PETRI-seq with second strand synthesis or template switch used for library preparation. Second-strand synthesis resulted in significantly more mRNAs per cell (p

    Techniques Used: Real-time Polymerase Chain Reaction, In Situ, Purification, Incubation, Polymerase Chain Reaction, Microscopy, Ligation, Sequencing

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    • dnase i reaction buffer  (New England Biolabs)
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    New England Biolabs dnase i reaction buffer
    Fluorescence image of fragmented DNA remaining after digestion by <t>DNase</t> I diffusing through microfluidic channels. Distance from reservoir inlet is 1.1mm.
    Dnase I Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fluorescence image of fragmented DNA remaining after digestion by DNase I diffusing through microfluidic channels. Distance from reservoir inlet is 1.1mm.

    Journal: bioRxiv

    Article Title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

    doi: 10.1101/2021.03.31.437857

    Figure Lengend Snippet: Fluorescence image of fragmented DNA remaining after digestion by DNase I diffusing through microfluidic channels. Distance from reservoir inlet is 1.1mm.

    Article Snippet: The buffer was either a 6-12:50 mixture (by volume) of 0.1M sodium hydroxide: 0.02M 2-(n-morpholino) ethanesulfonic acid (MES) or 1X NEB DNase I reaction buffer (NEB B0303S, 1X is 10mM Tris-HCl, 2.5mM MgCl2 , 0.5mM CaCl2).

    Techniques: Fluorescence

    DNA (at high density) fragmented on a surface by DNase I. Distance from inlet is 8.7mm.

    Journal: bioRxiv

    Article Title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

    doi: 10.1101/2021.03.31.437857

    Figure Lengend Snippet: DNA (at high density) fragmented on a surface by DNase I. Distance from inlet is 8.7mm.

    Article Snippet: The buffer was either a 6-12:50 mixture (by volume) of 0.1M sodium hydroxide: 0.02M 2-(n-morpholino) ethanesulfonic acid (MES) or 1X NEB DNase I reaction buffer (NEB B0303S, 1X is 10mM Tris-HCl, 2.5mM MgCl2 , 0.5mM CaCl2).

    Techniques:

    Schematic of stamping method for fragmenting surface-adsorbed. A PDMS stamp in the form of a grating is ‘inked’ with DNase I cuttting enzyme and is brought into contact with a surface on which DNA molecules have been deposited.

    Journal: bioRxiv

    Article Title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

    doi: 10.1101/2021.03.31.437857

    Figure Lengend Snippet: Schematic of stamping method for fragmenting surface-adsorbed. A PDMS stamp in the form of a grating is ‘inked’ with DNase I cuttting enzyme and is brought into contact with a surface on which DNA molecules have been deposited.

    Article Snippet: The buffer was either a 6-12:50 mixture (by volume) of 0.1M sodium hydroxide: 0.02M 2-(n-morpholino) ethanesulfonic acid (MES) or 1X NEB DNase I reaction buffer (NEB B0303S, 1X is 10mM Tris-HCl, 2.5mM MgCl2 , 0.5mM CaCl2).

    Techniques:

    Fluorescence image of SyBr Gold labeled DNA. Upper left area was covered with a solution containing 0.095U/μl of DNase I in NEB DNase I Reaction Buffer and shows effective digestion of DNA in that region.

    Journal: bioRxiv

    Article Title: Microfluidic delivery of cutting enzymes for fragmentation of surface-adsorbed DNA molecules

    doi: 10.1101/2021.03.31.437857

    Figure Lengend Snippet: Fluorescence image of SyBr Gold labeled DNA. Upper left area was covered with a solution containing 0.095U/μl of DNase I in NEB DNase I Reaction Buffer and shows effective digestion of DNA in that region.

    Article Snippet: The buffer was either a 6-12:50 mixture (by volume) of 0.1M sodium hydroxide: 0.02M 2-(n-morpholino) ethanesulfonic acid (MES) or 1X NEB DNase I reaction buffer (NEB B0303S, 1X is 10mM Tris-HCl, 2.5mM MgCl2 , 0.5mM CaCl2).

    Techniques: Fluorescence, Labeling

    Release of Pol II from chromatin via DNase I treatment. (A) Agarose gel (1%) showing the effect of different concentrations of DNAse I on DNA fragmentation. No difference is observed among treated conditions. (B) Western blot assessing Pol II release after treatment of 2×10 7 mouse ES cells with different Urea concentrations (0, 0.05 M, 0.25 M, 0.5 M) in the presence of 100U of DNase I. The conditions highlighted in red (0.05 M Urea 100U DNAse I) were used for the generation of all NET-prism libraries (P; Pellet, S; Supernatant).

    Journal: bioRxiv

    Article Title: NET-prism enables RNA polymerase-dedicated transcriptional interrogation at nucleotide resolution

    doi: 10.1101/246827

    Figure Lengend Snippet: Release of Pol II from chromatin via DNase I treatment. (A) Agarose gel (1%) showing the effect of different concentrations of DNAse I on DNA fragmentation. No difference is observed among treated conditions. (B) Western blot assessing Pol II release after treatment of 2×10 7 mouse ES cells with different Urea concentrations (0, 0.05 M, 0.25 M, 0.5 M) in the presence of 100U of DNase I. The conditions highlighted in red (0.05 M Urea 100U DNAse I) were used for the generation of all NET-prism libraries (P; Pellet, S; Supernatant).

    Article Snippet: 13| Add 100U of DNAse I in 100 µl of DNAse buffer (NEB) and place on ice for 20 min. Stop reaction by adding EDTA to a final concentration of 5 mM.

    Techniques: Agarose Gel Electrophoresis, Western Blot

    In situ assay specificity verified by DNase pretreatment. MT-CO1 sense DNA in situ hybridization assay on FFPE prostate tissues without RNase A ( A ), with RNase A ( B ), without DNase I ( C ), and with DNase I ( D ) pretreatments. Original magnification x40.

    Journal: bioRxiv

    Article Title: An in situ atlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells

    doi: 10.1101/2019.12.19.876144

    Figure Lengend Snippet: In situ assay specificity verified by DNase pretreatment. MT-CO1 sense DNA in situ hybridization assay on FFPE prostate tissues without RNase A ( A ), with RNase A ( B ), without DNase I ( C ), and with DNase I ( D ) pretreatments. Original magnification x40.

    Article Snippet: For DNase pretreatment, after steaming in Pretreatment II, the slides were treated with 100 μL DNase reaction buffer containing 10 μL DNase I Reaction Buffer (10X), 1 μL (2 units) DNAse I (M0303S, New England BioLabs, Ipswich, MA), and 89 μL nuclease-free H2 O.

    Techniques: In Situ, DNA In Situ Hybridization, Formalin-fixed Paraffin-Embedded