high capacity complementary dna reverse transcription kit  (Thermo Fisher)

Journal: Nucleic Acids Research

Article Title: Nanoscale dynamics of enhancer–promoter interactions during exit from pluripotency

doi: 10.1093/nar/gkaf1255

Figure Lengend Snippet: E–P distances of selected differentially expressed genes show small changes during differentiation from naive to primed mESCs. (A) Cell culture model of naive to primed transition in mESCs. (B) Changes in Nanog transcription levels (% GAPDH transcription) in naive and primed cells measured by qRT-PCR. The graph depicts median ± standard error of the mean ( n = 5 biological replicates). (C) Nanog genomic region for naive and primed cells, showing from top to bottom: DNA oligoFISH and NOVA FISH probes against promoter and selected enhancers, all predicted enhancers, connection of Nanog promoter to its enhancers, H3K27ac ChiP signal (from ), ATAC-seq signal (from ), CTCF binding motifs, and targeted enhancer regions (vertical gray stripes). Functionally validated enhancers are marked by ◆ . (D) Experimental workflow: regions of interest are marked with two-color DNA oligoFISH, the samples are imaged automatically with spinning disk confocal microscopy, FISH spots are detected automatically with subpixel localization accuracy and 3D distances between matched E–P spots are calculated to produce a E–P distance distribution of the population. Scale bar represents 1 μm. (E) 3D distance [μm] distributions between Nanog promoter and its −5, −45, +60, and +105 (putative) enhancers in naive and primed cells. Dashed line and number next to the histogram represent the median distance. The changes between naive and primed cells are not significant ( P > 0.05, two-sided Wilcoxon rank sum test, BH correction). From closest to furthest enhancer: n naive = 916, 1718, 837, 1220; n primed = 1037, 1038, 1362, 701; three biological replicates each. (F) Change in median 3D E–P distance [μm] of genes down- and up-regulated during the naive to primed transition. E–P pairs above the diagonal show increased distances during differentiation, while distance in pairs below the diagonal decreases. Each circle refers to a different enhancer. The test of statistical significance is the same as in panel (E). Functionally validated enhancers are marked by ◆ .

Article Snippet: Total RNA was isolated using NucleoSpin RNA, Mini kit for RNA purification (Marcherey-Nagel) according to the manufacturer’s instructions. cDNA was synthesized using the High-Capacity complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems) with 500 ng RNA as input. qRT-PCR with primers ( ) was performed in 10 μl reactions with 5 ng cDNA as input.

Techniques: Cell Culture, Quantitative RT-PCR, Binding Assay, Confocal Microscopy

Journal: Nucleic Acids Research

Article Title: Nanoscale dynamics of enhancer–promoter interactions during exit from pluripotency

doi: 10.1093/nar/gkaf1255

Figure Lengend Snippet: Shorter E–P distances correlate with active transcription. (A) Schematic representation: do enhancer and promoter come closer when the gene is actively transcribed? (B) Genomic location of Nanog intron RNA FISH probes. (C) Experimental workflow: intron RNA is marked by RNA FISH, imaged, promoters and enhancers are marked by DNA FISH, imaged, then RNA and DNA images are aligned using fiducial markers. (D) STED microscopy images (maximum intensity projections) of promoter (magenta), enhancers (yellow), and intron RNA (cyan) for Nanog . Scale bar represents 1 μm. (E) E–P 3D distance [µm] changes significantly based on distance of nascent RNA to P ( P < 0.05: *, P < 0.005: **, two-sided Wilcoxon rank sum test). n ≤0.25 = 46, n ≤0.5 = 156, n ≤1 = 272, n ≤1.5 = 172, n ≤3 = 259 over three biological replicates.

Article Snippet: Total RNA was isolated using NucleoSpin RNA, Mini kit for RNA purification (Marcherey-Nagel) according to the manufacturer’s instructions. cDNA was synthesized using the High-Capacity complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems) with 500 ng RNA as input. qRT-PCR with primers ( ) was performed in 10 μl reactions with 5 ng cDNA as input.

Techniques: Microscopy