cells direct one step qrt pcr kit  (Thermo Fisher)

Figure S1 and . (C) 3D-SIM images illustrating Shh -SBE6 separation in ESCs or in D7 NPCs. Scales bars are 5 μm (top two rows) and 1 μm (bottom, inset from center row). (D) Shh , Oct4 , and Nestin expression assayed by qRT-PCR during a time course of NPC differentiation. The graph shows mean (±SEM) log2 mRNA levels relative to Gapdh and normalized to the level in ESC (three technical replicates). (E) Violin plots showing Shh -SBE6 inter-probe distances in cell populations corresponding to the expression data in (D). (F) Kernel density plots showing Shh mRNA expression in single NPCs relative to Gapdh and normalized to the expression in ESC. Density is an arbitrary unit based on the frequency of the occurrence and the total counts and the size of the population (i.e., the binning of the data). Data from a biological replicate are shown in Figure S1 B. " title="... , Oct4 , and Nestin expression assayed by qRT-PCR during a time course of NPC differentiation. The ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Loss of Shh-Brain-Enhancer Proximity during Neuronal Differentiation (A) Map of the Shh regulatory domain showing the genes (black boxes), enhancers (green bars), and fosmid FISH probes (gray boxes). Probe and enhancer coordinates are listed in . (B) Violin plots showing the distribution of inter-probe distances (μm) between Shh and SBE6, SBE4, SBE2/3, ZRS, and CTRL probes in the nuclei of ESCs and D7 NPCs. Distances below the dotted horizontal line at 0.2 μm are considered co-localized. The asterisks on the FISH data represent Mann-Whitney U test significance between ESC and NPC populations. ∗∗ p < 0.01. Each violin plot represents one biological replicate; for other replicates and statistics, see Figure S1 and . (C) 3D-SIM images illustrating Shh -SBE6 separation in ESCs or in D7 NPCs. Scales bars are 5 μm (top two rows) and 1 μm (bottom, inset from center row). (D) Shh , Oct4 , and Nestin expression assayed by qRT-PCR during a time course of NPC differentiation. The graph shows mean (±SEM) log2 mRNA levels relative to Gapdh and normalized to the level in ESC (three technical replicates). (E) Violin plots showing Shh -SBE6 inter-probe distances in cell populations corresponding to the expression data in (D). (F) Kernel density plots showing Shh mRNA expression in single NPCs relative to Gapdh and normalized to the expression in ESC. Density is an arbitrary unit based on the frequency of the occurrence and the total counts and the size of the population (i.e., the binning of the data). Data from a biological replicate are shown in Figure S1 B.

Techniques Used: MANN-WHITNEY, Expressing, Quantitative RT-PCR

Figure Legend Snippet: Synthetic Activation of Shh and Increased Enhancer-Promoter Separation Using TALE-VP16 (A) Schematic of TALE-VP64 and TALE-VP128 constructs targeting the Shh promoter (tShh), SBE6, or SBE2. Repeat variable diresidue (RVD) code is displayed with one-letter abbreviations for amino acids. Self-cleaving (2A) peptide allows the expression of eGFP and cell isolation by fluorescence-activated cell sorting (FACS). A map of the targeting sites is shown at right. (B) Log2 mRNA levels of Shh, relative to Gapdh, assayed by qRT-PCR after TALE-VP64/128 expression in ESCs. Data show means (±SEMs) of three biological replicates normalized to ESCs expressing a control eGFP. (C) Violin plots representing one biological replicate of Shh-SBE6 inter-probe distances (μm) in ESCs expressing control eGFP, TALE-VP128 fusions targeting Shh promoter (tShh), SBE6, or SBE2. ∗∗ p < 0.01. Statistical data and replicate experiments are in . Distances below the dotted horizontal line at 0.2 μm are considered co-localized. (D) As in (C), but for VP64 recruitment to both SBE6 and SBE2 simultaneously (tSBE6+2), or a TALE with no fusion protein (tSBE6+2)-Δ. ∗∗ p < 0.01. Representative FISH images with probes for Shh (green) and SBE6 (red) in mESCs expressing tSBE(6+2)-VP64 and tSBE(6+2)-Δ are shown at right. (E) 5C heatmaps of the Shh regulatory region (chr5:28604000-29780000) with 16 kb binning and smoothing for ESCs and for ESCs expressing TALE-VP64 fusions targeting both SBE6 and SBE2. Difference 5C plots are shown above the main 5C plots. (F) Schematic representing TALE-LDB1 targeting sequences. (G) Three-color FISH with probes for Shh (green), SBE6 (magenta), and SBE2 (red) in mESCs expressing tShh-LDB1+tSBE2-LDB1. (H) Violin plots displaying Shh and SBE6 inter-probe distances (μm) in ESCs expressing eGFP or tShh-LDB1+tSBE6-LDB1. ∗∗ p < 0.01. (I) As in (H), but in cells expressing tShh-LDB1+tSBE2-LDB1. Shh-SBE6 distances are shown in the left-hand panel, and Shh-SBE2 distances are in the right-hand panel. ∗∗ p < 0.01. Statistical data relating to this figure are included in .

Techniques Used: Activation Assay, Construct, Expressing, Cell Isolation, Fluorescence, FACS, Quantitative RT-PCR

Figure Legend Snippet:

Techniques Used: Modification, Recombinant, Protease Inhibitor, Transfection, SYBR Green Assay, Staining, Whole Genome Amplification, Purification, Sequencing, Derivative Assay, Multiplex Assay, Software, Imaging, Microarray, Transformation Assay, RNA Sequencing Assay