Fig. 5: Two novel transcripts functionally involved in early embryogenesis.

a RT-PCR validation of Kdm4dl and XLOC_004958 during embryogenesis. The isoform structures of Kdm4dl and XLOC_004958 are shown in the middle; red arrows indicate the loci of the PCR primers, and the sizes of RT-PCR products are displayed. Sanger sequencing chromatograms of the RT-PCR products confirmed the splice junctions in the right panel. Representative data of two independent experiments is shown. b The relative expression of Kdm4dl and XLOC_004958 was measured by qPCR analysis. n = 2 biologically independent experiments. c Structural schematic showing the comparison of the previously annotated coding frame of Kdm4dl and the newly defined coding frame of Kdm4dl according to long-read data. In the lower panel, the newly defined protein sequence of Kdm4dl was compared with human KDM4E. Gray lines represent mismatched amino acids, white lines represent gaps, and light-yellow lines represent overlapping amino acids. d Diagram illustrating the CRISPR-mediated knockout of Kdm4dl and XLOC_004958; the scissors indicate the loci of the guide RNAs (sgRNA sequences are provided in Supplementary Table 3). e Morphological imaging of Kdm4dl and XLOC_004958 knockout embryos. The red arrows indicate abnormal blastocysts. The sgRNA-targeting GFP was defined as sgCtrl. Representative data of two independent experiments is shown. f The targeting efficiency of Kdm4dl and XLOC_004958 knockout embryos was calculated from Sanger sequencing results by TIDE (https://tide.deskgen.com/) and shown as the mean ± S.E.M. n = 3 embryos for Ctrl, n = 20 embryos for Kdm4dl, and n = 28 embryos for XLOC_004958. g The proportions of abnormal blastocysts following Kdm4dl and XLOC_004958 knockout in E4.5 embryos were calculated. n = 2 biologically independent experiments.