Extended Data Fig. 5: The Spocd1K464A mouse allele.
From: A nowhere-to-hide mechanism ensures complete piRNA-directed DNA methylation
a, Schematic representations of the mouse Spocd1 locus and encoded 1015 amino acid protein are shown, along with a schematic of the CRISPR targeting strategy showing the location of single-stranded oligo DNA donor (ssODN) and homology arms (HA) used. The sgRNA used for generation of the Spocd1K464A allele (red), adjacent PAM sites (red) and the primers used for genotyping (blue) are indicated, along with a representative sequencing trace of Spocd1K464A exon 7 harbouring the 3 bp mutation encoding the K464A mutation (highlighted in red). Sequencing was performed on n = 3 F1 animals. b, Representative image of PCR genotyping result for Spocd1+/+, Spocd1+/K464A and Spocd1K464A mice. PCR genotyping was performed for over 600 mice with similar results. c, Volcano plot showing enrichment (LFQ ratio of anti-SPOCD1 immunoprecipitates from wild-type/Spocd1K464A E16.5 foetal testis lysates) and statistical confidence (−log10(P-value of two-sided Student’s t-test)) of proteins co-purifying with wild-type SPOCD1 (right quadrant) or SPOCD1-K464A (left quadrant) (n = 3 with 24 foetal testes per replicate per genotype). All identified proteins meeting the enrichment cut-off are listed in Supplementary Table 2. d, Representative adult testis sections of n = 3 wild type, Spocd1K464A and Spocd1−/− stained in blue for DAPI and red for the DNA damage marker γH2AX (top) or apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (bottom). Scale bars, 50 μm.
