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Files in this Data Supplement:
Fig. S1. Expression levels of genes in the Kcnq1 domain in different cell types. Quantitative RT-PCR was carried out on TS, trophoblast giant cell, ES and embryoid body, embryo and placenta (E10.5) cDNA (n=6). 1 μl of 10× diluted cDNA was amplified using the primers in table 1 using SYBR Green I mastermix (Applied Biosystems). Where possible, primers were designed to span exons to prevent amplification of contaminating DNA. For each sample, the ΔCt value was calculated from the difference between the Ct value for the gene of interest and the housekeeping gene Gapdh. The ΔCt value was transformed (2 − ΔCt) to normalise them for statistical analysis. To represent relative expression levels graphically, the numbers are expressed relative to a wild-type trophoblast (E10.5). Normalisations with other housekeeping genes, such as 18S and Hprt, were also used to control for variability of normalisation genes. The results obtained were similar in all cases (data not shown). Cdkn1c, Kcnq1ot1 and Ascl2 increase significantly upon differentiation from TS to trophoblast giant cells. Phlda2, Cdkn1c, Tssc4 and Cd81 increase significantly upon differentiation from ES cells to embryoid bodies. Error bars represent s.e.m.
Fig. S2. Analysis of trophoblast giant cells from differentiated TS cells. (A) RT-PCR was used to analyse the allele-specific expression of genes within the Kcnq1 domain in C57BL/6J (B6)×Castaneus (Ca) trophoblast giant cells. The cells were differentiated for 6 days in the absence of FGF4 and heparin from TS cells. Maternal (M) and paternal (P) alleles were distinguished using RFLP or deletion/insertion polymorphisms described in the Materials and methods. For each gene a representative sample is shown with its corresponding reverse transcriptase negative control. Phlda2, Cdkn1c, Kcnq1ot1 and Ascl2 exhibit monoallelic expression and Tssc4 and Osbpl5 show biallelic expression. (B) ChIP assays were carried out on the giant cells described above to analyse allele-specific histone modifications with antibodies against H3Ac, H3K4me2, H3K9me2 and H3K27me3. The modifications associated with active chromatin regions are marked in black and dark grey, whereas those associated with repressive chromatin are marked in white and light grey. The parental alleles are distinguished by SNPs which are separated on SSCP gels. Phlda2, Cdkn1c and Kcnq1ot1 show an allelic bias in histone modifications (arrows) and Osbpl5, Tssc4, Cd81 and Ascl2 show no or little bias. Each panel is a representative example of the ChIP, beside it is a graphical representation of the ratio of the bound maternal allele to the bound paternal allele (normalised according to the input) shown for the active modifications and the ratio of paternal over maternal for repressive modification, except for Kcnq1ot1 where the ratios are reversed.
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