Etchevers:Cardiac morphogenesis/2008/10/02
 Cardiac morphogenesis
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ChIP-seq--- After the DGEmap meeting (see Conference notes), spoke with Asli about ChIP-seq on Illumina as agreed during HuDSEN proceedings. Meanwhile, Christelle had spoken with Swiss company to have prestation de service – looks economical. (As of September 5th, though not mentioned during my visit September 11th-13th, and my bringing Asli up to talk to Christelle seems to have pushed her to finally tell me about this enquiry.) Whole mushed embryo C16 4x clones in bacteria, do map to human genome. Had 10 μL of original 25μL from whole embryo with one quantity of Islet-1 antibody cf protocol. Bourgeons de membres also ChIP with Islet-1, do not amplify intronic sequence of FGF10 but could be useful for other targets. Pooled C16 membre inferieures: R625, R608 and R725. Remaining >22/25 μL. (Christelle uses 1 μL per 25 μL total volume of PCR). Of heart ChIP, used 11x C14 and C15 microdissected whole hearts. Remaining approx 15/25 μL. Ordre-of-magnitude thinking approx 0.5-1 ng per ChIP, so in the pg per μL range. -- Christelle has transfected luciferase construct into HeLa in 24 well plates. pGL-intron 1, intron 1 in sens, intron 1 beyond in antisense; pGL SV40 already a promoter that drives a basic light of luciferase. Nothing seems to have activated enhancer in the first trial, ratio of luciferase to other reporter is practically the same and the same if add the truncated form that used for EMSA and Western blot (pcDNA3.1). So this at least is transcribed. Fresh kit so assay itself is ok. The Islet added in did transactivate a wee bit (about 3x) but all numbers v v low. Proportion of plasmids out of 1 μg: can play with proportions. Did with 750 ng expression vector, 200 ng reporter with promoter construct, 50 ng CMV Renilla luciferase (other color) cf Celine chez Asma but the measure of NFKB activity is much more indirect before it gets back to the luciferase. However, Loic and Delphine for Phox2b and TCF4 used equivalent of 300ng and 600 ng. (260 ng vs 1.2 μg in 12-well dish). She will try the same early next week, 100+850+30μL, and 300+600+30μL. Each takes 8 wells of a 24-well plate.
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