dpnii  (New England Biolabs)


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    DpnII
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    DpnII 5 000 units
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    Restriction Enzymes
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    New England Biolabs dpnii
    DpnII
    DpnII 5 000 units
    https://www.bioz.com/result/dpnii/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    dpnii - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator"

    Article Title: Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026537

    Identification of mVIS. Strategy outline for identification of regions flanking DNA methylated viral integration sites (mVIS) within murine leukemias. Genomic DNA was digested with DpnII (step 1), followed by methylated DNA immunoprecipitation (MeDIP, step 2). MeDIP enriched fragments were ligated (step 3) and amplified using primers within the LTR (step 4). These fragments were hybridized on a DNA promoter array (step 5). Hypergeometric Analysis of Tiling Arrays (HAT) was used to identify regions flanking mVIS (step 6).
    Figure Legend Snippet: Identification of mVIS. Strategy outline for identification of regions flanking DNA methylated viral integration sites (mVIS) within murine leukemias. Genomic DNA was digested with DpnII (step 1), followed by methylated DNA immunoprecipitation (MeDIP, step 2). MeDIP enriched fragments were ligated (step 3) and amplified using primers within the LTR (step 4). These fragments were hybridized on a DNA promoter array (step 5). Hypergeometric Analysis of Tiling Arrays (HAT) was used to identify regions flanking mVIS (step 6).

    Techniques Used: Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Amplification, HAT Assay

    2) Product Images from "Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize"

    Article Title: Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000582

    Circular CRM1 RNAs induce chromatin loops in the centromere. (A) Anti-S9.6 RIP-qPCR was used to confirm the R-loop formation by 354-, 607-, and 277- to 296-nt circular CRM1 RNAs. Zm00001d007960 RNA was used as a negative control, and rRNA was used as a positive control. Chromatin-binding RNA was used for RIP. Actin was used as an internal reference gene. (B) Regions chosen for detecting the ssDNA sites are marked as 85–1, 253–1, 269–1, and 269–2. (C) ssDNA sites in CRM1 were checked using an S1 nuclease treatment of the nuclear DNA. DNA with no S1 nuclease treatment was used as a control template. The 607-left sequence was used as an internal reference gene. (D and E) Potential chromatin loops were induced by circular RNA inside a single CRM1 element (D) and between two CRM1 elements (E). Red, green, and yellow lines represent the 85-, 269-, and 253-bp regions, respectively. Black lines represent sequences on the left side of the 85-bp sequence and the right side of the 269-bp sequence. The blue ovals represent circular CRM1 RNAs. ①, ‘①’, ②, and ③ represent the broken ends on the two sides of the 253-bp sequence, the left side of the 85-bp sequence, and the right side of the 269-bp sequence. (F) 3C-PCR confirms the potential ligations of chromatin loops after DpnII digestion. The left panel shows the PCR results in the undigested, unligated samples and 3C samples under potential ligation forms. The right panel shows the sequences from the bands on the left, including the expected sequences, the first and the second part of the expected sequences, and the amplified sequences. (G and H) 3C-qPCR shows chromatin interactions inside a single CRM1 element (G) and between two CRM1 elements (H). The interaction frequencies between two DpnII -digested fragments were normalized to the 3C control template from the digested and ligated centromeric BAC clone and an internal reference gene, SAM . In (A), (C), (G), and (H), the columns and error bars represent the relative value and standard error of the means ( n = 3). In (A) and (C), the P values were determined using a Student t test: * P
    Figure Legend Snippet: Circular CRM1 RNAs induce chromatin loops in the centromere. (A) Anti-S9.6 RIP-qPCR was used to confirm the R-loop formation by 354-, 607-, and 277- to 296-nt circular CRM1 RNAs. Zm00001d007960 RNA was used as a negative control, and rRNA was used as a positive control. Chromatin-binding RNA was used for RIP. Actin was used as an internal reference gene. (B) Regions chosen for detecting the ssDNA sites are marked as 85–1, 253–1, 269–1, and 269–2. (C) ssDNA sites in CRM1 were checked using an S1 nuclease treatment of the nuclear DNA. DNA with no S1 nuclease treatment was used as a control template. The 607-left sequence was used as an internal reference gene. (D and E) Potential chromatin loops were induced by circular RNA inside a single CRM1 element (D) and between two CRM1 elements (E). Red, green, and yellow lines represent the 85-, 269-, and 253-bp regions, respectively. Black lines represent sequences on the left side of the 85-bp sequence and the right side of the 269-bp sequence. The blue ovals represent circular CRM1 RNAs. ①, ‘①’, ②, and ③ represent the broken ends on the two sides of the 253-bp sequence, the left side of the 85-bp sequence, and the right side of the 269-bp sequence. (F) 3C-PCR confirms the potential ligations of chromatin loops after DpnII digestion. The left panel shows the PCR results in the undigested, unligated samples and 3C samples under potential ligation forms. The right panel shows the sequences from the bands on the left, including the expected sequences, the first and the second part of the expected sequences, and the amplified sequences. (G and H) 3C-qPCR shows chromatin interactions inside a single CRM1 element (G) and between two CRM1 elements (H). The interaction frequencies between two DpnII -digested fragments were normalized to the 3C control template from the digested and ligated centromeric BAC clone and an internal reference gene, SAM . In (A), (C), (G), and (H), the columns and error bars represent the relative value and standard error of the means ( n = 3). In (A) and (C), the P values were determined using a Student t test: * P

    Techniques Used: Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Binding Assay, Sequencing, Polymerase Chain Reaction, Ligation, Amplification, BAC Assay

    3) Product Images from "Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex"

    Article Title: Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex

    Journal: Nature Communications

    doi: 10.1038/ncomms12743

    Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.
    Figure Legend Snippet: Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Techniques Used: Sequencing, Amplification, Real-time Polymerase Chain Reaction, Methylation, Polymerase Chain Reaction, Ligation, Injection, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay

    4) Product Images from "A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips"

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips

    Journal: Nature biotechnology

    doi: 10.1038/nbt.1716

    Scalable gene synthesis platform schematic for OLS Pool 2 Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip ( a ) and then cleaved to make a pool of oligonucleotides (b) . Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f) . Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.
    Figure Legend Snippet: Scalable gene synthesis platform schematic for OLS Pool 2 Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip ( a ) and then cleaved to make a pool of oligonucleotides (b) . Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f) . Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.

    Techniques Used: Synthesized, MicroChIP Assay, Polymerase Cycling Assembly, Clone Assay

    5) Product Images from "The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination"

    Article Title: The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination

    Journal: Nature

    doi: 10.1038/s41586-019-1547-y

    Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.
    Figure Legend Snippet: Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.

    Techniques Used: Activity Assay, Blocking Assay, Two Tailed Test

    6) Product Images from "Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress"

    Article Title: Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress

    Journal: Cell reports

    doi: 10.1016/j.celrep.2017.04.064

    Correction of C136G in MTTP rescues the ABL phenotype (A) Schematic strategy for correction of C136G in MTTP by CRISPR/Cas9. (B) iPSC from ABL patient was transfected with plasmids containing guide RNA and Cas9. Genomic DNA was extracted from GFP + colonies and subjected to PCR amplification. Subsequent DpnII digestion was applied to identify the positively targeted clones. (C) The corrected iPSC lines were tested for expression of pluripotency markers by real-time PCR. (D) Expression of hepatic genes was analyzed by real-time PCR in hepatocytes derived from the corrected iPSC lines. (E–G) Amount of newly synthesized apoB in the cell or secreted in the medium was measured at the end of a 2-hour label with [ 35 S]methionine/cysteine. F is a Western blot for apoB in the media. (H) Cellular lipid accumulation by Oil Red O staining following rescue of the C136G MTTP mutation by CRISPR/Cas9. Scale bar: 400 μm ± S.D. *P
    Figure Legend Snippet: Correction of C136G in MTTP rescues the ABL phenotype (A) Schematic strategy for correction of C136G in MTTP by CRISPR/Cas9. (B) iPSC from ABL patient was transfected with plasmids containing guide RNA and Cas9. Genomic DNA was extracted from GFP + colonies and subjected to PCR amplification. Subsequent DpnII digestion was applied to identify the positively targeted clones. (C) The corrected iPSC lines were tested for expression of pluripotency markers by real-time PCR. (D) Expression of hepatic genes was analyzed by real-time PCR in hepatocytes derived from the corrected iPSC lines. (E–G) Amount of newly synthesized apoB in the cell or secreted in the medium was measured at the end of a 2-hour label with [ 35 S]methionine/cysteine. F is a Western blot for apoB in the media. (H) Cellular lipid accumulation by Oil Red O staining following rescue of the C136G MTTP mutation by CRISPR/Cas9. Scale bar: 400 μm ± S.D. *P

    Techniques Used: CRISPR, Transfection, Polymerase Chain Reaction, Amplification, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Synthesized, Western Blot, Staining, Mutagenesis

    7) Product Images from "Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex"

    Article Title: Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex

    Journal: Nature Communications

    doi: 10.1038/ncomms12743

    Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.
    Figure Legend Snippet: Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Techniques Used: Sequencing, Amplification, Real-time Polymerase Chain Reaction, Methylation, Polymerase Chain Reaction, Ligation, Injection, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay

    8) Product Images from "Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay"

    Article Title: Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.973396

    MIAA identifies global influence of GC-content and differentially accessible motifs. A) GC-content observed to be correlated with accessibility in both stem and endoderm cells from positive (universally opening) and negative (universally closing) control sequences. B) GC-content correlated with accessibility in random DNA phrases. The regression model was trained on MIAA Dpn proportions with GC-content, replicate, and cell type-specific effects of 20 motifs and 26 motif pairs as features, and predicts well on (C) training data (n = 21,420) and (D) held-out test data (n = 4,404). The correlation reported is the Pearson correlation coefficient (r). E) Regression weights of individual motifs and motif pairs in stem and definitive endoderm cells. Hierarchical clustering of regression weights followed by motif enrichment recovers clusters representing cell type-specific transcription factor DNA binding motifs. F) Example of individual motifs (left, middle) which alone do not differentially open chromatin, but differentially open chromatin stem cells in combination (right). Each dot represents the average DpnII read proportion of an individual phrase, compared to shuffled controls (CTRL). Significance computed by paired t-test.
    Figure Legend Snippet: MIAA identifies global influence of GC-content and differentially accessible motifs. A) GC-content observed to be correlated with accessibility in both stem and endoderm cells from positive (universally opening) and negative (universally closing) control sequences. B) GC-content correlated with accessibility in random DNA phrases. The regression model was trained on MIAA Dpn proportions with GC-content, replicate, and cell type-specific effects of 20 motifs and 26 motif pairs as features, and predicts well on (C) training data (n = 21,420) and (D) held-out test data (n = 4,404). The correlation reported is the Pearson correlation coefficient (r). E) Regression weights of individual motifs and motif pairs in stem and definitive endoderm cells. Hierarchical clustering of regression weights followed by motif enrichment recovers clusters representing cell type-specific transcription factor DNA binding motifs. F) Example of individual motifs (left, middle) which alone do not differentially open chromatin, but differentially open chromatin stem cells in combination (right). Each dot represents the average DpnII read proportion of an individual phrase, compared to shuffled controls (CTRL). Significance computed by paired t-test.

    Techniques Used: Binding Assay

    Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).
    Figure Legend Snippet: Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Techniques Used: Synthesized, Methylation, Polymerase Chain Reaction, Amplification

    9) Product Images from "The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination"

    Article Title: The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination

    Journal: Nature

    doi: 10.1038/s41586-019-1547-y

    Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.
    Figure Legend Snippet: Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.

    Techniques Used: Activity Assay, Blocking Assay, Two Tailed Test

    10) Product Images from "Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing"

    Article Title: Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing

    Journal: bioRxiv

    doi: 10.1101/868117

    EPR-1 associates with H3K27 methylation in vivo and in vitro . a , Scatter plot showing the correlation of levels of H3K27me2/3 and GFP-EPR-1 WT for all genes (black dots), as determined by ChIP-seq. Line of best fit displayed in red (R 2 =0.8675). b , Western blot shows GFP-EPR-1 expression in the indicated strains. Phosphoglycerate kinase (PGK) is used as a loading control. Each genotype (except wild type, negative control) was run in biological duplicate and repeated. c , Quantification of the GFP band intensity averaged over 4 biological replicates. The intensities are relative to the corresponding PGK band and normalized to the wild-type average from the same blot. Filled bars represent the mean and error bars show standard deviation (a.u. signifies arbitrary units). d , Box and whisker plot of normalized GFP-EPR-1 ChIP-seq coverage from epr-1 WT , epr-1 PHD and epr-1 BAH strains is shown for the indicated regions of the genome. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. e , Box and whisker plot of normalized EPR-1 DamID-seq coverage from epr-1 WT , epr-1 PHD and epr-1 WT ; Δeed strains is shown for the indicated regions of the genome. Reads have been corrected for the frequency of GATC sites. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. f , DamID Southern blot of genomic DNA from the indicated strains digested with DpnI (DI), DpnII (DII) or left undigested (-). DpnII, which digests GATC sites without methylated adenines, reveals pattern of complete digestion in wild type. DpnI, which fails to digest GATC sites bearing adenine methylation, reveals the extent of methylation by Dam at probed regions ( NCU05173 and Tel VIIL, H3K27-methylated; his-3 , euchromatin). Ethidium bromide (EtBr) shows total DNA. Biological replicates are shown. g , An overlay of 1 H- 15 N HSQC spectra of 15 N-labelled HIS-SUMO-BAH EPR-1 fusion in the presence of increasing concentrations of H3K27me3 peptide. h , An overlay of 15 N-labelled HIS-SUMO-BAH EPR-1 fusion with the 15 N-labelled HIS-SUMO alone. Boxed areas are shown enlarged in Fig. 5d . Spectra are color coded as indicated.
    Figure Legend Snippet: EPR-1 associates with H3K27 methylation in vivo and in vitro . a , Scatter plot showing the correlation of levels of H3K27me2/3 and GFP-EPR-1 WT for all genes (black dots), as determined by ChIP-seq. Line of best fit displayed in red (R 2 =0.8675). b , Western blot shows GFP-EPR-1 expression in the indicated strains. Phosphoglycerate kinase (PGK) is used as a loading control. Each genotype (except wild type, negative control) was run in biological duplicate and repeated. c , Quantification of the GFP band intensity averaged over 4 biological replicates. The intensities are relative to the corresponding PGK band and normalized to the wild-type average from the same blot. Filled bars represent the mean and error bars show standard deviation (a.u. signifies arbitrary units). d , Box and whisker plot of normalized GFP-EPR-1 ChIP-seq coverage from epr-1 WT , epr-1 PHD and epr-1 BAH strains is shown for the indicated regions of the genome. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. e , Box and whisker plot of normalized EPR-1 DamID-seq coverage from epr-1 WT , epr-1 PHD and epr-1 WT ; Δeed strains is shown for the indicated regions of the genome. Reads have been corrected for the frequency of GATC sites. Box represents interquartile range, horizontal line is median, and whiskers represent minimum and maximum values. f , DamID Southern blot of genomic DNA from the indicated strains digested with DpnI (DI), DpnII (DII) or left undigested (-). DpnII, which digests GATC sites without methylated adenines, reveals pattern of complete digestion in wild type. DpnI, which fails to digest GATC sites bearing adenine methylation, reveals the extent of methylation by Dam at probed regions ( NCU05173 and Tel VIIL, H3K27-methylated; his-3 , euchromatin). Ethidium bromide (EtBr) shows total DNA. Biological replicates are shown. g , An overlay of 1 H- 15 N HSQC spectra of 15 N-labelled HIS-SUMO-BAH EPR-1 fusion in the presence of increasing concentrations of H3K27me3 peptide. h , An overlay of 15 N-labelled HIS-SUMO-BAH EPR-1 fusion with the 15 N-labelled HIS-SUMO alone. Boxed areas are shown enlarged in Fig. 5d . Spectra are color coded as indicated.

    Techniques Used: Methylation, In Vivo, In Vitro, Chromatin Immunoprecipitation, Western Blot, Expressing, Negative Control, Standard Deviation, Whisker Assay, Southern Blot

    11) Product Images from "Cleavage of DNA without loss of genetic information by incorporation of a disaccharide nucleoside"

    Article Title: Cleavage of DNA without loss of genetic information by incorporation of a disaccharide nucleoside

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg911

    Digestion pattern in the presence of 0.1 µg hybrid 3:4 ( A ) or 2:4 ( B ) and 1 or 10 U MboI or DpnII (indicated on top of phosphor image). The reaction temperature was 37°C and the reaction time 10 (lane 1), 30 (lane 2) or 120 min (lane 3).
    Figure Legend Snippet: Digestion pattern in the presence of 0.1 µg hybrid 3:4 ( A ) or 2:4 ( B ) and 1 or 10 U MboI or DpnII (indicated on top of phosphor image). The reaction temperature was 37°C and the reaction time 10 (lane 1), 30 (lane 2) or 120 min (lane 3).

    Techniques Used:

    Related Articles

    Clone Assay:

    Article Title: Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress
    Article Snippet: .. Then DNA was subjected to PCR amplification around the cutting site and subsequent DpnII (NEB) digest to analyze successfully edited clones. .. Oil Red O (Sigma O0625) and Nile Red (ThermoFisher) were used to label lipid droplets in iPSC-derived hepatocytes and cardiomyoyctes according to manufacturer’s instructions.

    Amplification:

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips
    Article Snippet: .. PCR amplification was followed by γ exonuclease digestion of 5′ phosphorylated strands, hybridization of the 3′ primer site to its complement, and cleavage of the 5′ and 3′ primer sites using USER enzyme mix and DpnII (New England Biolabs), respectively. .. Plate subpools were amplified from1 μL of OLS Pool 2 in 50 μL Phusion polymerase PCR reactions.

    Article Title: Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress
    Article Snippet: .. Then DNA was subjected to PCR amplification around the cutting site and subsequent DpnII (NEB) digest to analyze successfully edited clones. .. Oil Red O (Sigma O0625) and Nile Red (ThermoFisher) were used to label lipid droplets in iPSC-derived hepatocytes and cardiomyoyctes according to manufacturer’s instructions.

    Article Title: Chromatin state changes during neural development revealed by in vivo cell-type specific profiling
    Article Snippet: .. PCR adaptors were ligated to the cut DNA, before digestion with Dpn II (NEB) and PCR amplification (Clontech Advantage cDNA polymerase). .. Next-generation sequencing and data processing Following the DamID procedure, samples were sonicated in a Bioruptor Plus (Diagenode) to reduce the average DNA fragment size to 300 bp, and DamID adaptors were removed via either overnight Sau 3AI or Alw I digestion.

    Ligation:

    Article Title: The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination
    Article Snippet: .. DpnII or NlaIII was inactivated at 65°C for 20 min and samples were subjected to ligation under diluted condition with T4 DNA ligase (100 units, NEB, M0202L). ..

    Produced:

    Article Title: Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize
    Article Snippet: .. 3C in maize The 3C sample was produced according to a previously described method [ ], and the DNA was digested with the enzyme DpnII (NEB, Category Number R0543). ..

    Polymerase Chain Reaction:

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips
    Article Snippet: .. PCR amplification was followed by γ exonuclease digestion of 5′ phosphorylated strands, hybridization of the 3′ primer site to its complement, and cleavage of the 5′ and 3′ primer sites using USER enzyme mix and DpnII (New England Biolabs), respectively. .. Plate subpools were amplified from1 μL of OLS Pool 2 in 50 μL Phusion polymerase PCR reactions.

    Article Title: Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress
    Article Snippet: .. Then DNA was subjected to PCR amplification around the cutting site and subsequent DpnII (NEB) digest to analyze successfully edited clones. .. Oil Red O (Sigma O0625) and Nile Red (ThermoFisher) were used to label lipid droplets in iPSC-derived hepatocytes and cardiomyoyctes according to manufacturer’s instructions.

    Article Title: Chromatin state changes during neural development revealed by in vivo cell-type specific profiling
    Article Snippet: .. PCR adaptors were ligated to the cut DNA, before digestion with Dpn II (NEB) and PCR amplification (Clontech Advantage cDNA polymerase). .. Next-generation sequencing and data processing Following the DamID procedure, samples were sonicated in a Bioruptor Plus (Diagenode) to reduce the average DNA fragment size to 300 bp, and DamID adaptors were removed via either overnight Sau 3AI or Alw I digestion.

    Methylated DNA Immunoprecipitation:

    Article Title: Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator
    Article Snippet: .. MeDIP Ten µg genomic DNA was digested overnight with 100 U of DpnII (New England Biolabs, Ipswich, MA, USA). .. Four µg digested DNA was denatured for 10′ at 95°C and incubated with either 2.5 µg anti-5-methylcytidine (BI-MECY-1000, Eurogentec, Liège, Belgium) or mouse pre-immune IgG (Sigma-Aldrich, Zwijndrecht, The Netherlands) in 500 µL IP-buffer (PBS with 0.05% Triton X-100) for 2 hrs at 4°C, followed by incubation with 30 µL of washed beads (M-280 sheep-anti-mouse IgG, Invitrogen, San Diego, CA, USA) for 2 hrs at 4°C.

    Hybridization:

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips
    Article Snippet: .. PCR amplification was followed by γ exonuclease digestion of 5′ phosphorylated strands, hybridization of the 3′ primer site to its complement, and cleavage of the 5′ and 3′ primer sites using USER enzyme mix and DpnII (New England Biolabs), respectively. .. Plate subpools were amplified from1 μL of OLS Pool 2 in 50 μL Phusion polymerase PCR reactions.

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    New England Biolabs dpnii
    Identification of mVIS. Strategy outline for identification of regions flanking DNA methylated viral integration sites (mVIS) within murine leukemias. Genomic DNA was digested with <t>DpnII</t> (step 1), followed by methylated DNA immunoprecipitation <t>(MeDIP,</t> step 2). MeDIP enriched fragments were ligated (step 3) and amplified using primers within the LTR (step 4). These fragments were hybridized on a DNA promoter array (step 5). Hypergeometric Analysis of Tiling Arrays (HAT) was used to identify regions flanking mVIS (step 6).
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    Identification of mVIS. Strategy outline for identification of regions flanking DNA methylated viral integration sites (mVIS) within murine leukemias. Genomic DNA was digested with DpnII (step 1), followed by methylated DNA immunoprecipitation (MeDIP, step 2). MeDIP enriched fragments were ligated (step 3) and amplified using primers within the LTR (step 4). These fragments were hybridized on a DNA promoter array (step 5). Hypergeometric Analysis of Tiling Arrays (HAT) was used to identify regions flanking mVIS (step 6).

    Journal: PLoS ONE

    Article Title: Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator

    doi: 10.1371/journal.pone.0026537

    Figure Lengend Snippet: Identification of mVIS. Strategy outline for identification of regions flanking DNA methylated viral integration sites (mVIS) within murine leukemias. Genomic DNA was digested with DpnII (step 1), followed by methylated DNA immunoprecipitation (MeDIP, step 2). MeDIP enriched fragments were ligated (step 3) and amplified using primers within the LTR (step 4). These fragments were hybridized on a DNA promoter array (step 5). Hypergeometric Analysis of Tiling Arrays (HAT) was used to identify regions flanking mVIS (step 6).

    Article Snippet: MeDIP Ten µg genomic DNA was digested overnight with 100 U of DpnII (New England Biolabs, Ipswich, MA, USA).

    Techniques: Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Amplification, HAT Assay

    Enrichment of 3T3-HOX11 DPs by cDNA RDA. (A) Ethidium-stained agarose gel electrophoresis of starting cDNA Dpn II fragment representations and various RDA enriched populations obtained by mutual subtraction of NIH 3T3 cells (3T3) with 3T3-HOX11 (left lanes show products obtained with NIH 3T3 cells as the tester and right lanes show products obtained with 3T3-HOX11 cells as the tester). Tester representation (R; unselected cDNA) and DP1, DP2, DP3, and DP4 are shown. The arrow indicates an enriched HOX11 Dpn II fragment identified by Southern filter hybridization with a HOX11 -specific probe. (B) Southern filter hybridizations (with Slim1 , Aldh1 , HOX11 , and β- actin probes as indicated) of the agarose gel-fractionated RDA DPs shown in panel A. Fragment sizes are indicated. (C) Northern filter hybridization of 10 μg of total RNA prepared from untransfected NIH 3T3 cells and three independent 3T3-HOX11 clones and hybridized with HOX11 , Slim1 , Aldh1 , and ATP synthase probes (as indicated). 3T3-HOX11 clones 5 and 18 were found to express HOX11 protein, while clone 11 did not (data not shown). Hybridization of the filter with ATP synthase was used to assess the quality of the RNA transferred.

    Journal: Molecular and Cellular Biology

    Article Title: The T-Cell Oncogenic Protein HOX11 Activates Aldh1 Expression in NIH 3T3 Cells but Represses Its Expression in Mouse Spleen Development

    doi:

    Figure Lengend Snippet: Enrichment of 3T3-HOX11 DPs by cDNA RDA. (A) Ethidium-stained agarose gel electrophoresis of starting cDNA Dpn II fragment representations and various RDA enriched populations obtained by mutual subtraction of NIH 3T3 cells (3T3) with 3T3-HOX11 (left lanes show products obtained with NIH 3T3 cells as the tester and right lanes show products obtained with 3T3-HOX11 cells as the tester). Tester representation (R; unselected cDNA) and DP1, DP2, DP3, and DP4 are shown. The arrow indicates an enriched HOX11 Dpn II fragment identified by Southern filter hybridization with a HOX11 -specific probe. (B) Southern filter hybridizations (with Slim1 , Aldh1 , HOX11 , and β- actin probes as indicated) of the agarose gel-fractionated RDA DPs shown in panel A. Fragment sizes are indicated. (C) Northern filter hybridization of 10 μg of total RNA prepared from untransfected NIH 3T3 cells and three independent 3T3-HOX11 clones and hybridized with HOX11 , Slim1 , Aldh1 , and ATP synthase probes (as indicated). 3T3-HOX11 clones 5 and 18 were found to express HOX11 protein, while clone 11 did not (data not shown). Hybridization of the filter with ATP synthase was used to assess the quality of the RNA transferred.

    Article Snippet: Double-stranded cDNA (approximately 2 μg) was digested with Dpn II (NEB), extracted with phenol-chloroform, and ethanol precipitated.

    Techniques: Staining, Agarose Gel Electrophoresis, Hybridization, Northern Blot, Clone Assay

    Linx -Related Topological Features Are Not Implicated in Xist Regulation (A) The Linx locus, CTCF binding, and orientation of CTCF motifs associated with CTCF chromatin immunoprecipitation sequencing (ChIP-seq) peaks. Orientation of CTCF motifs within the Tsix-TAD is represented above. The targeted deletions ΔLinxCBS (∼25 kb) and ΔLinx-int1 (∼51 kb) are indicated. See STAR Methods for sources of CTCF, DNaseI, and H3K27Ac datasets. (B and C) 5C profiles of the Tsix-TAD (B) and the two Xic TADs (C); pooled data from two biological replicates for each genotype. Differential map is corrected for deletion (see STAR Methods ). Gray pixels represent either the deleted region or filtered contacts. (D) Left: cross used for analysis of RNA allelic ratios in female hybrid embryos. Right: Xist RNA allelic ratios; each black dot corresponds to a single female embryo. Statistical analysis was performed using a two-tailed t test. The table summarizes the number of embryos collected. Analysis of Atp7a RNA allelic ratios and reverse cross is shown in Figures S5 A and S5B. (E and F) Capture-C profiles for LinxP (E) and Xist (F) viewpoints, at different time points of differentiation of XX (Pgk12.1) mESCs. Data represent one replicate; two or three replicates for each time point were performed and are identical to the one shown (data available in GEO). Profiles represent number of contacts for each DpnII fragment per 10,000 total contacts within a specified region (see STAR Methods ). CTCF ChIP-seq on male mESCs is represented below ( Nora et al., 2017 ). (G–I) 5C differential maps for mutant male mESCs: ΔLinxE (G), ΔLinxP (H) and LinxP-inv (I); pooled data from two biological replicates for each genotype. 5C profiles for each genotype are shown in Figure S5 D. Gray pixels correspond to either deleted regions or filtered contacts. (J) Quantification of 5C inter-TAD contacts (see Figure S5 E for details). Bars represent the average of the calculated proportions of four (E14 and ΔLinxP) or two (ΔLinxE and LinxP-inv) independent replicates. Statistical analysis was performed using a two-tailed t test ( ∗∗ p

    Journal: Molecular Cell

    Article Title: A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary

    doi: 10.1016/j.molcel.2019.10.030

    Figure Lengend Snippet: Linx -Related Topological Features Are Not Implicated in Xist Regulation (A) The Linx locus, CTCF binding, and orientation of CTCF motifs associated with CTCF chromatin immunoprecipitation sequencing (ChIP-seq) peaks. Orientation of CTCF motifs within the Tsix-TAD is represented above. The targeted deletions ΔLinxCBS (∼25 kb) and ΔLinx-int1 (∼51 kb) are indicated. See STAR Methods for sources of CTCF, DNaseI, and H3K27Ac datasets. (B and C) 5C profiles of the Tsix-TAD (B) and the two Xic TADs (C); pooled data from two biological replicates for each genotype. Differential map is corrected for deletion (see STAR Methods ). Gray pixels represent either the deleted region or filtered contacts. (D) Left: cross used for analysis of RNA allelic ratios in female hybrid embryos. Right: Xist RNA allelic ratios; each black dot corresponds to a single female embryo. Statistical analysis was performed using a two-tailed t test. The table summarizes the number of embryos collected. Analysis of Atp7a RNA allelic ratios and reverse cross is shown in Figures S5 A and S5B. (E and F) Capture-C profiles for LinxP (E) and Xist (F) viewpoints, at different time points of differentiation of XX (Pgk12.1) mESCs. Data represent one replicate; two or three replicates for each time point were performed and are identical to the one shown (data available in GEO). Profiles represent number of contacts for each DpnII fragment per 10,000 total contacts within a specified region (see STAR Methods ). CTCF ChIP-seq on male mESCs is represented below ( Nora et al., 2017 ). (G–I) 5C differential maps for mutant male mESCs: ΔLinxE (G), ΔLinxP (H) and LinxP-inv (I); pooled data from two biological replicates for each genotype. 5C profiles for each genotype are shown in Figure S5 D. Gray pixels correspond to either deleted regions or filtered contacts. (J) Quantification of 5C inter-TAD contacts (see Figure S5 E for details). Bars represent the average of the calculated proportions of four (E14 and ΔLinxP) or two (ΔLinxE and LinxP-inv) independent replicates. Statistical analysis was performed using a two-tailed t test ( ∗∗ p

    Article Snippet: Digestion was performed overnight by adding 50 μL of DpnII (Capture-C) or HindIII (5C) buffer and 10 μL of high-concentration DpnII or HindIII (NEB) and incubating samples at 37°C in a thermomixer.

    Techniques: Binding Assay, ChIP-sequencing, Chromatin Immunoprecipitation, Two Tailed Test, Capture-C, Mutagenesis

    Circular CRM1 RNAs induce chromatin loops in the centromere. (A) Anti-S9.6 RIP-qPCR was used to confirm the R-loop formation by 354-, 607-, and 277- to 296-nt circular CRM1 RNAs. Zm00001d007960 RNA was used as a negative control, and rRNA was used as a positive control. Chromatin-binding RNA was used for RIP. Actin was used as an internal reference gene. (B) Regions chosen for detecting the ssDNA sites are marked as 85–1, 253–1, 269–1, and 269–2. (C) ssDNA sites in CRM1 were checked using an S1 nuclease treatment of the nuclear DNA. DNA with no S1 nuclease treatment was used as a control template. The 607-left sequence was used as an internal reference gene. (D and E) Potential chromatin loops were induced by circular RNA inside a single CRM1 element (D) and between two CRM1 elements (E). Red, green, and yellow lines represent the 85-, 269-, and 253-bp regions, respectively. Black lines represent sequences on the left side of the 85-bp sequence and the right side of the 269-bp sequence. The blue ovals represent circular CRM1 RNAs. ①, ‘①’, ②, and ③ represent the broken ends on the two sides of the 253-bp sequence, the left side of the 85-bp sequence, and the right side of the 269-bp sequence. (F) 3C-PCR confirms the potential ligations of chromatin loops after DpnII digestion. The left panel shows the PCR results in the undigested, unligated samples and 3C samples under potential ligation forms. The right panel shows the sequences from the bands on the left, including the expected sequences, the first and the second part of the expected sequences, and the amplified sequences. (G and H) 3C-qPCR shows chromatin interactions inside a single CRM1 element (G) and between two CRM1 elements (H). The interaction frequencies between two DpnII -digested fragments were normalized to the 3C control template from the digested and ligated centromeric BAC clone and an internal reference gene, SAM . In (A), (C), (G), and (H), the columns and error bars represent the relative value and standard error of the means ( n = 3). In (A) and (C), the P values were determined using a Student t test: * P

    Journal: PLoS Biology

    Article Title: Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize

    doi: 10.1371/journal.pbio.3000582

    Figure Lengend Snippet: Circular CRM1 RNAs induce chromatin loops in the centromere. (A) Anti-S9.6 RIP-qPCR was used to confirm the R-loop formation by 354-, 607-, and 277- to 296-nt circular CRM1 RNAs. Zm00001d007960 RNA was used as a negative control, and rRNA was used as a positive control. Chromatin-binding RNA was used for RIP. Actin was used as an internal reference gene. (B) Regions chosen for detecting the ssDNA sites are marked as 85–1, 253–1, 269–1, and 269–2. (C) ssDNA sites in CRM1 were checked using an S1 nuclease treatment of the nuclear DNA. DNA with no S1 nuclease treatment was used as a control template. The 607-left sequence was used as an internal reference gene. (D and E) Potential chromatin loops were induced by circular RNA inside a single CRM1 element (D) and between two CRM1 elements (E). Red, green, and yellow lines represent the 85-, 269-, and 253-bp regions, respectively. Black lines represent sequences on the left side of the 85-bp sequence and the right side of the 269-bp sequence. The blue ovals represent circular CRM1 RNAs. ①, ‘①’, ②, and ③ represent the broken ends on the two sides of the 253-bp sequence, the left side of the 85-bp sequence, and the right side of the 269-bp sequence. (F) 3C-PCR confirms the potential ligations of chromatin loops after DpnII digestion. The left panel shows the PCR results in the undigested, unligated samples and 3C samples under potential ligation forms. The right panel shows the sequences from the bands on the left, including the expected sequences, the first and the second part of the expected sequences, and the amplified sequences. (G and H) 3C-qPCR shows chromatin interactions inside a single CRM1 element (G) and between two CRM1 elements (H). The interaction frequencies between two DpnII -digested fragments were normalized to the 3C control template from the digested and ligated centromeric BAC clone and an internal reference gene, SAM . In (A), (C), (G), and (H), the columns and error bars represent the relative value and standard error of the means ( n = 3). In (A) and (C), the P values were determined using a Student t test: * P

    Article Snippet: 3C in maize The 3C sample was produced according to a previously described method [ ], and the DNA was digested with the enzyme DpnII (NEB, Category Number R0543).

    Techniques: Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Binding Assay, Sequencing, Polymerase Chain Reaction, Ligation, Amplification, BAC Assay