Journal: Genome Research

Article Title: ATAC-seq reveals regional differences in enhancer accessibility during the establishment of spatial coordinates in the Drosophila blastoderm

doi: 10.1101/gr.242362.118

Figure Lengend Snippet: Domain-restricted ATAC-seq profiling along antero-posterior axis in the blastoderm embryo. ( A ) Selected AP domains are targeted by expressing a nuclear tag, UNC84-3×FLAG, under control of well-characterized enhancers of gap and pair-rule genes. All reporter constructs are integrated at the same genomic site (attP2) to standardize genetic background. After homogenization of staged embryos (cellularizing blastoderm, stage 5, 2:50–3:10 h after egg laying), tagged nuclei are affinity-purified with anti-FLAG antibodies, followed by Tn5 transposase fragmentation and ATAC-seq library preparation. An ATAC-seq library representing an entire pool of nuclei from homogenized embryos (whole-embryo) serves as a control. ( B ) Overview of the tagged domains (D1–D7). Selected embryos immunolabeled with an anti-FLAG antibody show spatially restricted expression domains of the nuclear tag. Each domain is additionally schematized (green bars) to indicate its position along the AP axis. Embryos are positioned with anterior to the left and dorsal side up.

Article Snippet: UNC84-3×FLAG nuclear marker was generated by C-terminal fusion of unc-84 coding sequence (amplified from pMUH_unc84_ tdTFlag, a gift from Sean Eddy; Addgene plasmid #46024) ( ) to a 25-aa peptide linker (HL4 from ) followed by a 3×FLAG tag (Sigma).

Techniques: Expressing, Control, Construct, Homogenization, Affinity Purification, Immunolabeling

Journal: Genome Research

Article Title: ATAC-seq reveals regional differences in enhancer accessibility during the establishment of spatial coordinates in the Drosophila blastoderm

doi: 10.1101/gr.242362.118

Figure Lengend Snippet: Regional differences in chromatin accessibility. Accessibility profiles of individual tagged domains and a whole-embryo control at the locus of giant ( gt ), a gene of the AP patterning network of the gap class. Tracks show normalized coverage of 1- to 100-bp ATAC-seq fragments, smoothed over a sliding window of 15 bp. AP positions of the profiled domains are indicated schematically on the left (green shading). Blue bars and underlying shaded regions indicate coordinates of known giant enhancers (REDfly names and references provided in Supplemental Table S8 ). Spatial activity of each enhancer in blastoderm embryos is illustrated above (RNA in situ hybridization of a reporter gene) (reprinted from ). Note that gt_(-2)_broad was used as a driver of UNC84-3×FLAG in D7, and the additional copy in attP2 partially contributes to its elevated ATAC-seq signal (as discussed in Supplemental Methods ). Genomic coordinates and gene models: FlyBase Release 5.57 .

Article Snippet: UNC84-3×FLAG nuclear marker was generated by C-terminal fusion of unc-84 coding sequence (amplified from pMUH_unc84_ tdTFlag, a gift from Sean Eddy; Addgene plasmid #46024) ( ) to a 25-aa peptide linker (HL4 from ) followed by a 3×FLAG tag (Sigma).

Techniques: Control, Activity Assay, RNA In Situ Hybridization

Journal: Ecology and Evolution

Article Title: Sea star populations diverge by positive selection at a sperm-egg compatibility locus

doi: 10.1002/ece3.487

Figure Lengend Snippet: (a) Schematic diagram of bindin gene organization and coding sequence structure. (a) Main diagram (above) shows the 914-amino acid haplotype with the modal number of collagen-like copies and tandem repeats. Insets below show the extra collagen-like copy with 3' flanking region, and the extra tandem repeat, each found only once in our sample. Rectangles with solid black and grey borders show tandem repeat copies that are present in all alleles, dashed rectangles show tandem repeats that vary in copy number among alleles. Asterisk shows location of the single recombination site at codon site 855, black triangle denotes the 1285 bp intron at codon site 856, and solid black rectangle denotes the 135 bp invariant core. Colors in sequence are the default colors used in Se-al (v.2.0) (cyan = H, R, K; magenta = G, P, T, A, S; black = E, Q, D, N; green = L, V, M, I; blue = Y). (b) Map of study region and location of two main population samples. Dashed gray line represents location of phylogeographic break identified by Keever et al. .

Article Snippet: Sea star bindin includes a short core region similar to the invariant core domain of bindin in sea urchins, but most of the sea star bindin coding sequence is structurally distinctive from sea urchins and consists of several types of long repetitive motifs ( ; see also Patiño et al. ).

Techniques: Sequencing

Journal: Annals of Translational Medicine

Article Title: microRNA-1 inhibits cardiomyocyte proliferation in mouse neonatal hearts by repressing CCND1 expression

doi: 10.21037/atm.2019.08.68

Figure Lengend Snippet: Ability of miR-1 to target the 3'UTR of IGF1 and CCND1 mRNA in neonatal cardiomyocytes. (A) Alignment of 5’ seeding region within miR-1 mature sequence and the putative binding sites within the 3'UTR region of IGF1, CCND1, Frs2 and FoxP1-mRNAs using TargetScan database. (B) Construct luciferase reporter containing the predicted binding sites within the 3'UTR of IGF1, CCND1, Frs2 and FoxP1 mRNAs into downstream of the firefly luciferase gene of pmirGLO vector. (C) Dual-Glo Luciferase reporter assays revealed reduced luciferase activity after co-transfection of pmirGLO-IGF1 3'UTR vector and pmirGLO-CCND1 3'UTR vector, respectively, with miR-1 mimics. The results were compared with luciferase activities from cardiomyocytes transfected with miR-1 mimics N.C. and pmirGLO empty vectors. The data are presented as mean ± SD from triplicated experiments. **, P<0.01 vs. miR-1 mimics NC or pmirGLO empty vectors.

Article Snippet: The amplified fragments were individually subcloned upstream of the luciferase coding sequence of Firefly/Renilla dual reporter vector pmirGLO (Promega, Madison, USA).

Techniques: Sequencing, Binding Assay, Construct, Luciferase, Plasmid Preparation, Activity Assay, Cotransfection, Transfection