Journal: Development (Cambridge, England)
Article Title: H3K9 tri-methylation at Nanog times differentiation commitment and enables the acquisition of primitive endoderm fate
doi: 10.1242/dev.201074
Figure Lengend Snippet: The lack of H3K9me3 at Nanog leads to delayed differentiation. (A) Log2 Nanog mRNA levels measured by RT-qPCR and normalised to Tbp during EB differentiation in the indicated cell lines. Each dot represents an independent replicate and the line the corresponding mean with s.e.m. Expression differences between E14Tg2a and ΔK9 clones were evaluated at each day of differentiation with unpaired one-tailed Student's t -tests ( P =0.002143, 0.0485, 0.06709, 7.367e−06 for days 0, 4, 6 and 8, respectively). (B) PCA of 16,336 transcripts quantified by RNA-seq (TPM >1 in at least one sample) in wild-type (E14Tg2a, black; n =3) and ΔK9 (red and orange; n =3 for each) cells undergoing EB differentiation for the indicated days. (C) Heatmap of 4100 transcripts displaying gene expression changes during EB differentiation [FDR<0.05 and abs(log2FC)>1 in at least one comparison to undifferentiated cells; n =3 for each cell line], clustered by k-means after z -score normalisation. Left: Relative gene expression ( z- score) in wild-type (E14Tg2a) and ΔK9 cells during EB differentiation. Right: Corresponding log2FC of each developmental stage indicated at the top versus E4.5 epiblast, as previously reported . (D) Boxplot (median; 25-75% percentiles; 1.5× the inter-quartile range) of the log2FC shown in C for each cluster. Cluster C6 is highlighted in orange. The highest distribution of log2FC for PRE E4.5 for C6 with respect to the other clusters was assessed with a Kolmogorov–Smirnov test ( P <1e−07 for all comparisons). d, days; ECT, ectoderm; END, endoderm; EPI: epiblast; MES, mesoderm; PRE, primitive endoderm.
Article Snippet: Stranded, poly-A-selected RNA-seq libraries were prepared and sequenced (paired-end 150 bp reads; around 50 million each) by Novogene UK.
Techniques: Quantitative RT-PCR, Expressing, Clone Assay, One-tailed Test, RNA Sequencing Assay
![The lack of H3K9me3 at Nanog leads to delayed differentiation. (A) Log2 Nanog mRNA levels measured by RT-qPCR and normalised to Tbp during EB differentiation in the indicated cell lines. Each dot represents an independent replicate and the line the corresponding mean with s.e.m. Expression differences between E14Tg2a and ΔK9 clones were evaluated at each day of differentiation with unpaired one-tailed Student's t -tests ( P =0.002143, 0.0485, 0.06709, 7.367e−06 for days 0, 4, 6 and 8, respectively). (B) PCA of 16,336 transcripts quantified by <t>RNA-seq</t> (TPM >1 in at least one sample) in wild-type (E14Tg2a, black; n =3) and ΔK9 (red and orange; n =3 for each) cells undergoing EB differentiation for the indicated days. (C) Heatmap of 4100 transcripts displaying gene expression changes during EB differentiation [FDR<0.05 and abs(log2FC)>1 in at least one comparison to undifferentiated cells; n =3 for each cell line], clustered by k-means after z -score normalisation. Left: Relative gene expression ( z- score) in wild-type (E14Tg2a) and ΔK9 cells during EB differentiation. Right: Corresponding log2FC of each developmental stage indicated at the top versus E4.5 epiblast, as previously reported . (D) Boxplot (median; 25-75% percentiles; 1.5× the inter-quartile range) of the log2FC shown in C for each cluster. Cluster C6 is highlighted in orange. The highest distribution of log2FC for PRE E4.5 for C6 with respect to the other clusters was assessed with a Kolmogorov–Smirnov test ( P <1e−07 for all comparisons). d, days; ECT, ectoderm; END, endoderm; EPI: epiblast; MES, mesoderm; PRE, primitive endoderm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2333/pmc09482333/pmc09482333__develop-149-201074-g4.jpg)