Fig. 2
An in vitro screen for H3K9 methyltransferases involved in the maintenance of XCI identifies Setdb1. a MEFs were transduced with a pool of shRNA retroviruses and a DNA sample taken before 5-aza treatment (Additional file 3: Figure S3). After treatment, DNA was extracted from the GFP+ and GFP− populations obtained by FACS. Hairpin sequences were amplified by PCR with a common forward primer and barcoded reverse primers. b The most significantly differentially represented hairpins (p < 0.1) with a positive fold change in the GFP+ sample compared to the GFP− sample. Dnmt1 positive control hairpins are highlighted in orange and Setdb1 hairpins in blue. c Immortalised X XistΔAa X GFPi MEFs were transduced with validated H3K9 methyltransferase shRNAs, treated with 5-aza and GFP reactivation assessed by FACS as shown in Figure S1B. n = 4, mean + s.e.m, one-way ANOVA with Dunnett correction for multiple testing, and only H3K9 methyltransferase shRNAs were included in the comparison. **p < 0.01; ****p < 0.001. d Bright field and GFP fluorescence images of representative E7.5 Setdb1 +/+, Setdb1 +/gt, and Setdb1 gt/gt X XistΔAa X GFPi embryos at 63 × magnification. Embryonic (em), extra-embryonic (ex) and ectoplacental cone (ec) tissues are indicated. Scale bar represents 200 µm. See also Additional files 3: Figure S3, 5: Figure S4, 4: Table S1, 6: Figure S5 and 7: Figure S6