Extended Data Figure 9. Generation of the Spocd1^HA mouse allele.

a, Schematic representation of the SPOCD1 protein and Spocd1 locus as well as design of the sgRNA targeting the 3’ UTR near the translation termination site on Spocd1 exon 15. The Spocd1^HA allele encodes for a carboxy-terminal GGGGS linker followed by the HA epitope tag. The protospacer adjacent motif (PAM) site was mutated to inhibit re-targeting of the Spocd1^HA allele by the sgRNA-CAS9 complex. All inserted nucleotides and corresponding encoded amino acids are highlighted in red. The SPOCD1-HA protein is shown as a schematic representation. b, Schematic representation of the targeting strategy to generate the Spocd1^HA allele with a short single stranded DNA oligo donor (ssODN) of 200 nucleotides containing 5’ and 3’ homology arms (5’HA and 3’HA) of 72 nucleotides. c, Representative image of genotyping results for Spocd1^+/+ , Spocd1^HA/+ and Spocd1^HA/HA animals. Similar results were obtained for all animals of the Spocd1^HA line. d, Sequencing trace of part of a PCR amplicon of the HA epitope tag insertion site from a Spocd1^HA/HA animal. The experiment was repeated with identical results on n=2 animals. e, f, g, Representative images of wildtype (e), Spocd1^HA/+ (f) and Miwi2^HA/+ (g) testis sections at the indicated developmental time point probed with anti-HA antibody in green. DNA stained with DAPI in blue. Scale bars, 10 μm. The representative images presented in panels e to g are from experiments done n=3 mice as biological replicates with similar results.