DEV031328 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The Kcnq1ot1 transcript spans 120 kb. Strand-specific RT-PCR on total RNA from E13.5 wild-type (C57BL6/JOlaHsd) and KvDMR1^+/- embryos upstream, within and downstream of Kcnq1ot1. The same results were obtained independently from two embryos of each genotype. NP indicates a no primer control; + and − indicate reactions with and without reverse transcriptase, respectively. The detection of transcription from +300bp to +120.4 kb in the direction of Kcnq1ot1 in the wild-type embryo but not in the KvDMR1^+/- embryo provides evidence that Kcnq1ot1 spans 120 kb. No Kcnq1ot1 transcription was detected beyond the position of the polyadenylated tail, suggesting this is the endpoint of transcription.

• Supplemental Figure S2 -

  Fig. S2. Kcnq1ot1 is transcribed by Pol II and has a half-life of 3.4 hours. NIH 3T3 cells were treated with 5 µg/ml α-amanitin for the indicated times and expression levels were determined by qRT-PCR. (A) Expression levels were normalised to an untreated sample and represented as mean±s.e.m. (n=4 for all time points except 2 and 4 hours, where n=8). cmyc is a positive control, as the mRNA has a short half-life and is transcribed by Pol II. My1 and 18S rRNA are negative controls as they are transcribed by RNA polymerases III and I, respectively. (B) The expression data were log-transformed and mRNA half-lives were taken by calculating the inverse of the slope of the fitted regression line.