r0543  (New England Biolabs)

Journal: bioRxiv

Article Title: Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

doi: 10.1101/2020.11.17.386797

Figure Lengend Snippet: Nucleolar-DamID identifies NADs A. Chromosomal view of NADs, LADs ( Peric-Hupkes et al., 2010 ), A and B compartments ( Dalcher et al., 2020 ), and early and late replicating DNA ( Marchal et al., 2018 ) in ESCs. NADs are measured as log 2 ratio of m6A levels between H2B-Dam-NoLS and H2B-Dam. iNAD: regions not contacting the nucleolus. iLAD: regions not contacting the NL. Chromosomes 2 and 18 are shown. B. NAD coverage. Bars represent NAD coverage values of each mouse chromosome. Red lines showed NAD coverage values in the centromere-proximal end of each chromosome. Chromosome bearing rRNA genes are depicted with orange numbers and bars. Dotted line shows NAD whole genome coverage. C. Nucleolar H2B-Dam chromosomal interaction maps. NAD-only and NAD/LAD regions were represented with orange and grey colors, respectively. Chromosome bearing rRNA genes are depicted with orange numbers. D. Genomic contacts with rRNA genes (rDNA contacts) obtained from published HiC maps ( Bonev et al., 2017 ). Data represent the proportion of identified unique contacts for each chromosome. E. Representative images showing normalized count score of rDNA contacts obtained from the HiC-rDNA on chromosome 9, which does not harbor rRNA genes, and chromosome 18, which contains rRNA genes. F. Distribution of rDNA contacts for each chromosome. Values represent the proportion of the number of unique contacts for each chromosome quintile. Statistical significance ( P -values) for the experiments was calculated using the unpaired two-tailed t-test (****

Article Snippet: In order to cut unmethylated GATC sequences, DNA was digested for 3h at 37°C with Dpn II (New England Biolabs) and heat inactivated at 65°C for 20 minutes.

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

doi: 10.1101/2020.11.17.386797

Figure Lengend Snippet: Distinct layers of repressive chromatin states distinguish genomic domains according to their interaction with the nucleolus, nuclear lamina, or both A. Venn diagram showing the proportion of NAD-only and NAD/LAD regions in NADs identified by Nucleolar-DamID. B. Venn diagram showing the proportion of LAD-only and NAD/LAD regions in LADs of ESCs. C. Lamin B1-DamID scores plotted over NAD-only and NAD/LAD boundaries in ESCs. D. Gene density of total genome, NAD subclasses, and LAD-only. E. Amounts (%) of NAD-only, LAD-only and NAD/LAD in A and B compartments. F. Amounts (%) of early and late replicating DNA of NAD-only, LAD-only and NAD/LAD sequences. G. Expression values (RPKM) of genes within A compartment (A Comp.), NAD-only, LAD-only, and NAD/LAD. Statistical significance ( P -values) was calculated using the unpaired two-tailed t-test (***

Article Snippet: In order to cut unmethylated GATC sequences, DNA was digested for 3h at 37°C with Dpn II (New England Biolabs) and heat inactivated at 65°C for 20 minutes.

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

doi: 10.1101/2020.11.17.386797

Figure Lengend Snippet: Nucleolar-DamID in ESCs A. Pearson correlation of Nucleolar-DamID experiments in ESCs and NPCs. B. Violin plot showing distribution of NAD length. C. Chromosomal view of NADs in ESCs. NADs are measured as log 2 ratio of m6A levels between H2B-Dam-NoLS vs. H2B-Dam. iNAD: inter-NAD regions. iLAD: inter-LAD regions. Pink bars correspond to sequence found in nucleolar hubs using SPRITE method ( Quinodoz et al., 2018 ). D. Upper panel. NAD and LAD composition of chromosome 14 and FISH probe corresponding to a strong LAD (blue bar). Lower panel. Representative images from immunofluorescence for nucleolin (green) combined with DNA FISH using probes corresponding to LAD-only region of chromosome 14 (red), and DAPI (blue). The signal of the LAD probe was located close to NL in the majority of analyzed ESCs (27/33).

Article Snippet: In order to cut unmethylated GATC sequences, DNA was digested for 3h at 37°C with Dpn II (New England Biolabs) and heat inactivated at 65°C for 20 minutes.

Techniques: Sequencing, Fluorescence In Situ Hybridization, Immunofluorescence

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

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

Journal: bioRxiv

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

doi: 10.1101/2020.03.02.973396

Figure Lengend 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.

Article Snippet: Genomic DNA library preparation was performed by performing DpnI and DpnII digests using the following conditions: DpnI digest: 10 ug genomic DNA + 10 uL CutSmart Buffer (New England Biolabs) + 2 uL DpnI (New England Biolabs) + up to 100 uL water.

Techniques: Binding Assay

Journal: bioRxiv

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

doi: 10.1101/2020.03.02.973396

Figure Lengend 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).

Article Snippet: Genomic DNA library preparation was performed by performing DpnI and DpnII digests using the following conditions: DpnI digest: 10 ug genomic DNA + 10 uL CutSmart Buffer (New England Biolabs) + 2 uL DpnI (New England Biolabs) + up to 100 uL water.

Techniques: Synthesized, Methylation, Polymerase Chain Reaction, Amplification