Journal: eLife

Article Title: Losing Dnmt3a dependent methylation in inhibitory neurons impairs neural function by a mechanism impacting Rett syndrome

doi: 10.7554/eLife.52981

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Article Snippet: Sectioned were mounted in VECTASHIELD HardSet Antifade Mounting Medium without DAPI (Vector Laboratories H-1400) and let dry overnight at 4°C.

Techniques: Labeling, Protease Inhibitor, Lysis, Plasmid Preparation, Electron Microscopy, Western Blot, Antibody Labeling, Purification, Methylation, Lambda DNA Preparation, Software

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation

doi: 10.3389/fcimb.2018.00235

Figure Lengend Snippet: Eap binding to DNA is affected by the state and type of DNA. Eap (0.5 μg/ml) was co-incubated with DNA samples (100 ng/ml) obtained from different sources, and subsequently attached to OTS silicon prior to AFM imaging. Representative AFM height images of Eap/DNA complexes adhering to OTS silicon from three independent experiments are shown: (A) Phage lambda DNA. (B) Artificial 1.4 kb PCR DNA-product. (C) Pst I-digested plasmid pBR322 (D) Circular pBR322 isolated from E. coli DH5α. (E) Sheared herring sperm DNA. Eap molecules putatively adhering to DNA (white arrows), OTS (yellow arrows), or cross-linking DNA (green arrows) are indicated. DNA molecules displayed in (A–C) , and (E) were aligned to OTS silicon by DNA combing.

Article Snippet: Herring sperm DNA (10164142), phage lambda DNA (D3779), and pBR322 (N3033L) DNA were purchased from Invitrogen (Karlsruhe, Germany), Sigma-Aldrich, and New England Biolabs (Frankfurt, Germany), respectively.

Techniques: Binding Assay, Incubation, Imaging, Lambda DNA Preparation, Plasmid Preparation, Isolation

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , Schematic of the construction of CRISPR screen library. A CRISPR knockout plasmid library containing sgRNA-barcode associations was constructed to generate a lentivirus library, which was transduced into human A549-Cas9 cells to produce a cell library. b , Design of the CRISPR screen plasmid and the lentiviral integration strategy. The sgRNA-barcode cassette was composed of human U6 promoter (hU6, blue), sgRNA (yellow), barcode (purple) and UMI (dark blue) sequences and placed within 3’ long terminal repeat (3’ LTR, dark gray), downstream of a strong RNA Pol II promoter (CMV, green). This cassette was duplicated and inserted within 5’ LTR (light gray) during lentiviral integration. Therefore, the cassette within 5’ LTR was able to express the sgRNA for genome editing, while the other copy was driven by CMV promoter to express a high level of barcode RNA for BARC-FISH decoding. Other elements on the plasmid including EF-1α promoter (pink), Puromycin resistance gene (magenta) and WPRE element (brown) were shown. c , BARC-FISH decoding efficiency in the CRISPR screen cell library. After BARC-FISH decoding procedure, the decoded barcodes were compared and matched to the barcodes determined by NGS. 33% of the imaged cells contained barcodes with perfect matches. After the error correction, 51% of the cells contained matched barcodes. d , Analysis of barcode quality determined by NGS. The sgRNA-barcode associations in the CRISPR screen cell library were determined by NGS (see , “ ” section). In total, 4,469 barcodes were detected, among which 76% were good codes associating with one unique sgRNA. 413 sgRNAs targeting 137 genes and 8 non-targeting controls were found to be associated with these good codes. 412 of the 413 sgRNAs were observed in the image-based screen.

Article Snippet: The GFP lentiviral overexpression vector, pLenti-GFP, was purchased from OriGene (OriGene, PS100093).

Techniques: CRISPR, Knock-Out, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , The barcode plasmid library was assembled from individual oligos through overlapping ligation, overlapping PCR, limited-cycle PCR and Gibson Assembly (see , “ ” section). Each of the forward-strand oligos contained three alternative sequences (in the smaller gray dashed box), represented by three different colors cyan, magenta, and yellow. The overlapping oligos in the reverse strand contained 9 alternative sequences (in the larger gray dashed box). Oligos at the two ends carried PCR priming regions (straight gray lines). The barcode was divided into two halves which were subjected to overlapping ligation to form two double-stranded fragments. The two fragments were assembled by overlapping PCR to form a full-length barcode. The barcode was then amplified and added with UMI (unique molecular identifier) by limited-cycle PCR primers. The barcode-UMI fragments were inserted into a digested plasmid backbone through Gibson Assembly to construct the final barcode plasmid library. To clone the CRISPR screen plasmid library, sgRNA fragments and barcode-UMI fragments were amplified from the premade sgRNA plasmid library and barcode plasmid library respectively, through limited-cycle PCR. The sgRNA and barcode-UMI were then Gibson Assembled into a digested lentiviral plasmid backbone to generate the final CRISPR screen plasmid library (see , “ ” section). The UMI was necessary for sequencing-based mapping of barcode-sgRNA associations (see , “ ”). b , Percentage of frameshift mutations of sgCHD7, sgTBX6, sgRUNX3, sgNIPBL and sgLRIF1. c , Two representative cells from the screen datasets were shown to demonstrate the Geminin staining strategy for G1 phase cell detection. Geminin antibody stain (magenta) is absent in a G1 phase cell (left), which showed two DNA FISH foci of TAD3 (yellow) of chr22. The S/G2 phase cell (right) is positive for Geminin stain and have four DNA FISH foci of TAD3 in two pairs, indicating replicated TAD3 DNA. Because Geminin and the yellow-green fiducial beads were imaged using the same laser channel, bead patterns were seen in both images (small, round magenta spots outside of the nuclei). Scale bar: 10 μm.

Article Snippet: The GFP lentiviral overexpression vector, pLenti-GFP, was purchased from OriGene (OriGene, PS100093).

Techniques: Plasmid Preparation, Ligation, Amplification, Construct, CRISPR, Sequencing, Staining

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , Log2 fold change of inter-TAD distance of siCHD7 compared to siCtrl. Number of traces analyzed: 3,558 (siCtrl) and 4,134 (siCHD7). b , Log2 fold change of short-range (defined as spatial distances between genomic regions that are less than 3Mb apart) and long-range (defined as spatial distances between genomic regions that are more than 3Mb apart) inter-TAD distances between siCHD7 and siCtrl. c , Log2 fold change of inter-TAD distance of CHD7 overexpression compared to GFP overexpression. Number of traces analyzed: 3,157 (GFP OE) and 1,174 (CHD7 OE). d , Log2 fold change of short-range and long-range inter-TAD distances between CHD7 and GFP overexpression. e , Log2 fold change of inter-TAD distance of TSA-treated cells compared to DMSO-treated cells. Number of traces analyzed: 1,214 (DMSO) and 2,223 (TSA). f , Log2 fold change of short-range and long-range inter-TAD distances between cells with TSA and DMSO treatment. g , Log2 fold change of inter-TAD distance of CHD7-ΔBRK (BRK domain deletion) overexpression compared to GFP overexpression. Number of traces analyzed: 3,415 (CHD7-ΔBRK OE) and 2,164 (GFP OE). h , Log2 fold change of short-range and long-range inter-TAD distances between CHD7-ΔBRK OE and GFP OE. i , Log2 fold change of inter-TAD distance of CHD7-K999R overexpression compared to GFP overexpression. Number of traces analyzed: 2,045 (CHD7-K999R) and 2,164 (GFP OE) j , Log2 fold change of short-range and long-range inter-TAD distances between CHD7-K999R OE and GFP OE. All chromatin tracing experiments in this figure were done in the A549 cell background, targeting chr22. P values were calculated by two-sided Wilcoxon signed rank test. k , Representative images of dye-labeled lambda DNA without/with purified CHD7. Scale bar: 500 μm. l , Spatial distribution of 130 genes decoded by RNA MERFISH in siCtrl and siCHD7 cells. Two representative cells are shown for each condition. Scale bar: 10 μm. m , Average RNA counts per cell for each gene in siCHD7 versus siCtrl cells. The red dashed line represents the x=y line. n , −Log10 false discovery rate (FDR) versus log2 fold change (log2fc) of average RNA counts per cell for each gene from siCtrl to siCHD7. Number of cells analyzed: 1,979 (siCtrl) and 1,186 (siCHD7) in m and n . o , Representative cell images of poly-A stain for siCtrl and siCHD7 cells. Scale bar: 20 μm. p , Mean fluorescent intensity of poly-A stain in individual nuclei of siCtrl and siCHD7 cells. P value was calculated by two-sided Wilcoxon rank sum test. Number of nuclei analyzed: 666 (siCtrl) and 594 (siCHD7).

Article Snippet: The GFP lentiviral overexpression vector, pLenti-GFP, was purchased from OriGene (OriGene, PS100093).

Techniques: Over Expression, Labeling, Lambda DNA Preparation, Purification, Staining

Journal: bioRxiv

Article Title: Perturb-tracing enables high-content screening of multiscale 3D genome regulators

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: a , A-B compartment profile of chr22 in A549-Cas9 cells with GFP overexpression. b , A-B compartment profile of chr22 in A549-Cas9 cells with CHD7 overexpression. c , Polarization indices of cells with GFP (white) and CHD7 (orange) overexpression and the corresponding randomized controls (shadowed). d , Compartmental contact frequencies of cells with GFP of CHD7 (shadowed) overexpression in A compartments (red), across A and B compartments (purple) and in B compartments (blue). e , Overall inter-TAD distance of chr22 in cells with GFP and CHD7 overexpression. f , Radii of gyration of chr22 in cells with GFP and CHD7 overexpression. P values in c , d and f were calculated by two-sided Wilcoxon rank sum test. P value in e were calculated by two-sided Wilcoxon signed rank test.

Article Snippet: The GFP lentiviral overexpression vector, pLenti-GFP, was purchased from OriGene (OriGene, PS100093).

Techniques: Over Expression

Journal: eLife

Article Title: Human RPA activates BLM’s bidirectional DNA unwinding from a nick

doi: 10.7554/eLife.54098

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Article Snippet: Recombinant DNA reagent , pTWIN1 (plasmid) , New England BioLabs , N6951S , Expression of core BLM in E. coli.

Techniques: Recombinant, Lambda DNA Preparation, Plasmid Preparation, Expressing, DNA Purification, Staining, Software

Journal: STAR Protocols

Article Title: Visualizing DNA replication by single-molecule analysis of replicated DNA

doi: 10.1016/j.xpro.2023.102721

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Article Snippet: Anti-avidin antibody, biotinylated (0.5 g /mL) (dilution 1:15) , Vector Laboratories , Cat #M0392S.

Techniques: Plasmid Preparation, Recombinant, Avidin-Biotin Assay, Marker, Lambda DNA Preparation, Saline, Agarose Gel Electrophoresis, Software, Hybridization