Dataset 2.tar

Zipped; raw ABI sequence data files

Additional file 2: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Tutorial for the bioinformatics including sgRNA/primer deisgn and MiSeq analysis. (PDF 7778 kb

Additional file 4: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Amplicon screening data used for Fig. 4a-c. (TXT 142 kb

Additional file 5: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Results from KASP genotyping used for Fig. 4d. (TXT 910 bytes

Additional file 7: of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Code used to produce the plot in Figs. 4 and 5, Additional file 3: Figure S1. (TXT 14 kb

Additional file 3: Figure S1 and Figure S2. of Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish

Comparison of induced indels between germline and somatic tissues. (a) Plot shows the same data as Fig. 4a, split into separate plots for each sgRNA. (b) Plot of frequencies for individual variants in sperm versus fin clip (same data as in Fig. 4b with full axes). (PDF 374 kb

zfsnp_map_zv9

zfsnp_map_zv

zfsnp_ped_zv9_dryad

zfsnp_ped_zv9_drya

Canonical Wnt signaling is downregulated in <i>fto</i> morphants zebrafish.

<p>(A) Dual luciferase assay using the β-catenin responsive TopFlash construct shows loss of reporter assay activity in <i>fto</i>MO embryos analysed at both 24 hpf (con: 1.000 SEM ±0.073, ftoMO: 0.491 SEM ±0.105) and 48 hpf (con:1.000 SEM ±0.103, ftoMO 0.580 SEM ±0.066) stages. (B) <i>Lef1</i> transcripts, a canonical Wnt target gene, were analysed by <i>in situ</i> hybridisation (ISH) at 48 hpf. <i>Fto</i> morphants showed marked loss of <i>lef1</i> expression in the optic-tectum (arrows). Scale bar: 500 µm. n =  con 56/66, <i>fto</i>MO 40/53. (C) Loss of β-Catenin was confirmed in <i>fto</i> morphants by western blotting at 48 hpf. β-Catenin protein levels were quantified relative to the loading control (Actin). (D) ISH analysis of <i>ctnnb1</i> (zebrafish <i>β-catenin 1</i>) at 48 hpf showed upregulation of transcripts specifically in areas of the lateral hindbrain (arrowheads). Scale bar: 500 µm. n =  con 70/70, <i>fto</i>MO 65/68 (E) <i>Fto</i> morphant <i>Tg(7xTCF-Xla.Siam:GFP)^ia4</i> display loss of GFP accumulation in both the Telen- and Diencephalic regions of the brain when compared to uninjected controls at 48 hpf, embryos viewed from a dorsal perspective. Scale bar: 100 µm; n =  con 20/20, <i>fto</i>MO 18/20.</p

β-Catenin dependant canonical Wnt signaling is compromised in Fto deficient cells.

<p>(A) Cytoplasmic, membrane and nuclear fractions of control (<i>Fto^+/+</i>) and Fto knockout (<i>Fto^−/−</i>) MEFs treated with control (−) or Wnt3a -conditioned medium (+) for 4 hours were analysed by Western blot using β-Catenin antibody. Hsp90 and c-Jun were used as loading controls for cytoplasmic and nuclear fractions, respectively. (B) <i>Fto</i> mRNA expression in control and <i>Fto</i> knockout MEFs as determined by RT Real Time PCR. The data shown represent the mean ±SEM, (n = 3) (C) Quantification of cytoplasmic, membrane and nuclear β-catenin by ELISA in control and <i>Fto</i> knockout MEFs treated with control or Wnt3a -conditioned medium for 4 hours. The data shown represent the mean ±SEM, (n = 5). One-way ANOVA with Tukey’s multiple comparison test was performed, ***P<0.05, NS: not significant. (D) Immunofluorescence of control and <i>Fto</i> knockout MEFs treated with control and Wnt3a conditioned medium for 4 hours. Scale bar indicates 20 µm. This image is representative of three separate experiments.</p