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Nucleosome regulatory dynamics in response to TGFβ.

Enroth S, Andersson R, Bysani M, Wallerman O, Termén S, Tuch BB, De La Vega FM, Heldin CH, Moustakas A, Komorowski J, Wadelius C - Nucleic Acids Res. (2014)

Bottom Line: We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci.We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci.Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing.

View Article: PubMed Central - PubMed

Affiliation: The Linnaeus Centre for Bioinformatics, Biomedical Center, Uppsala University, SE-75124 Uppsala, Sweden.

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(A) Coverage plots of RNA-seq data over the SMAD7 gene. The TGFβ stimulated (TGFB+) data have been normalized to the same sequencing depth as the unstimulated (TGFB−). (B) Scatter plot of RPKM-counts over genes for TGFB+ and TGFB−. The genes selected for Taqman validation are marked with black triangles. (C) Taqman validation results for 25 selected genes. The RNA-seq is represented as log2 of sequence depth normalized fold change between TGFB+ and TGFB−, and the Taqman values are also log2 of fold change between TGFB+ and TGFB−.
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Figure 8: (A) Coverage plots of RNA-seq data over the SMAD7 gene. The TGFβ stimulated (TGFB+) data have been normalized to the same sequencing depth as the unstimulated (TGFB−). (B) Scatter plot of RPKM-counts over genes for TGFB+ and TGFB−. The genes selected for Taqman validation are marked with black triangles. (C) Taqman validation results for 25 selected genes. The RNA-seq is represented as log2 of sequence depth normalized fold change between TGFB+ and TGFB−, and the Taqman values are also log2 of fold change between TGFB+ and TGFB−.

Mentions: RNA from HepG2 cells with and without TGFβ1-stimulation was isolated as described above. The RNA was treated with DNase I (Qiagen) to degrade any genomic DNA. Reverse transcription was performed with 1.0 μg of RNA per 20-μl reaction using the iScript complementary DNA (cDNA) Synthesis Kit (Bio-Rad). Triplicate 25-μl reactions were prepared containing 1.0 μl of cDNA, TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan Gene Expression Assay Mix (Applied Biosystems) specific for the transcript investigated according to the instructions of the manufacturer. PCR was performed on an Applied Biosystems 7000 Real-Time PCR System (Applied Biosystems) with SDS software 1.2.3, using the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Control reactions to demonstrate the specificity of the reactions were for each gene expression assay: use of cDNA synthesized without reverse transcriptase and without template RNA, respectively, and replacing the cDNA with water altogether. All controls passed and are not included in the figure. Expression levels were determined with the comparative Ct method using GAPDH as reference, related to the RNA sequencing expression levels and presented in Figure 8C.


Nucleosome regulatory dynamics in response to TGFβ.

Enroth S, Andersson R, Bysani M, Wallerman O, Termén S, Tuch BB, De La Vega FM, Heldin CH, Moustakas A, Komorowski J, Wadelius C - Nucleic Acids Res. (2014)

(A) Coverage plots of RNA-seq data over the SMAD7 gene. The TGFβ stimulated (TGFB+) data have been normalized to the same sequencing depth as the unstimulated (TGFB−). (B) Scatter plot of RPKM-counts over genes for TGFB+ and TGFB−. The genes selected for Taqman validation are marked with black triangles. (C) Taqman validation results for 25 selected genes. The RNA-seq is represented as log2 of sequence depth normalized fold change between TGFB+ and TGFB−, and the Taqman values are also log2 of fold change between TGFB+ and TGFB−.
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Related In: Results  -  Collection

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Figure 8: (A) Coverage plots of RNA-seq data over the SMAD7 gene. The TGFβ stimulated (TGFB+) data have been normalized to the same sequencing depth as the unstimulated (TGFB−). (B) Scatter plot of RPKM-counts over genes for TGFB+ and TGFB−. The genes selected for Taqman validation are marked with black triangles. (C) Taqman validation results for 25 selected genes. The RNA-seq is represented as log2 of sequence depth normalized fold change between TGFB+ and TGFB−, and the Taqman values are also log2 of fold change between TGFB+ and TGFB−.
Mentions: RNA from HepG2 cells with and without TGFβ1-stimulation was isolated as described above. The RNA was treated with DNase I (Qiagen) to degrade any genomic DNA. Reverse transcription was performed with 1.0 μg of RNA per 20-μl reaction using the iScript complementary DNA (cDNA) Synthesis Kit (Bio-Rad). Triplicate 25-μl reactions were prepared containing 1.0 μl of cDNA, TaqMan Gene Expression Master Mix (Applied Biosystems) and TaqMan Gene Expression Assay Mix (Applied Biosystems) specific for the transcript investigated according to the instructions of the manufacturer. PCR was performed on an Applied Biosystems 7000 Real-Time PCR System (Applied Biosystems) with SDS software 1.2.3, using the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Control reactions to demonstrate the specificity of the reactions were for each gene expression assay: use of cDNA synthesized without reverse transcriptase and without template RNA, respectively, and replacing the cDNA with water altogether. All controls passed and are not included in the figure. Expression levels were determined with the comparative Ct method using GAPDH as reference, related to the RNA sequencing expression levels and presented in Figure 8C.

Bottom Line: We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci.We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci.Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing.

View Article: PubMed Central - PubMed

Affiliation: The Linnaeus Centre for Bioinformatics, Biomedical Center, Uppsala University, SE-75124 Uppsala, Sweden.

Show MeSH