Supplementary Figure 4: Zebrafish models for Smarcd2 deficiency.
(a) Knockdown of smarcd2 by the splice-blocking morpholino oligonucleotides was examined by RT–PCR. Morpholino SB2 (MO SB2) failed to disrupt smarcd2 splicing. Top, amplification of smarcd2 exon boundaries; bottom, β-actin control; (+) and (–) indicate samples prepared with and without reverse transcriptase, i.e., monitoring for genomic DNA contamination. Image cropped from one gel. The full-length gel image is shown in Supplementary Data 3b. (b) Fluorescence image of Danio rerio strain Tg(mpx:EGFP)i114, control versus smarcd2-morphant embryo. Reduced numbers of GFP-expressing neutrophils are observed in morphant versus control fish embryos. Acquired images: CTRL (n = 15 images), ATG MO (n = 15 images), SB1 MO (n = 15 images) and SB2 MO (n = 15 images). (c) Comprehensive analysis of neutrophil numbers in Tg(mpx:EGFP)i114 at 72 h.p.f. after injection of morpholino oligonucleotides (nonspecific control versus translation-start-site blocker (ATG) and splice-site blocker (SB1 and SB2) MOs against smarcd2. Numbers represent fluorescence-labeled neutrophils per individual fish embryos. Data were pooled from two independent morpholino experiments: CTRL n = 15, ATG n = 15, SB1 n = 15, SB2 n = 15 fish. Center value, mean; error bars, s.d. P values were calculated by two-tailed unpaired t test. Replicates: 2. Please compare to Supplementary Tables 19 and 20. These results are an independent verification of the data in Figure 3b in another neutrophil reporter line. (d) Sanger chromatogram of the CRISPR/Cas9-edited frameshift allele smarcd21/1 (1/1) versus the smarcd2 wild-type allele (wt) indicating the induced mutation c.66dup (Mut).
