Figure 1. Validation of H2A.Z hypervariant-specific RNAi. A, H2A.Z.1 and H2A.Z.2 amino acid sequences are depicted where three different amino acid residues are indicated in red. The target sequences of shH2afz and shH2afv are noted in blue. B, left, Representative images of smFISH of H2afv and H2afz in E18 rat cortex. Right, Quantification of H2afz and H2afv molecules; particle count per 212 μm2. N = 3, *p < 0.05 (one-tailed unpaired t test). Scale bar, 10 μm. C, Cortical neurons in culture were infected with lentiviruses delivering either shH2afz or shH2afv and total RNA was collected after indicated number of days. Compared to control neurons, changes in H2afz and H2afv mRNA levels were determined by qPCR, normalized to Gapdh mRNA levels (internal control) and are depicted here as fold change. NS, not significant. *p < 0.05. N = 3–6. D, Timeline of infection and treatment for all assays performed in dissociated cortical neurons. Cells were infected with lentiviruses between 5 and 7 d after plating (gray line), and assays were performed between 4 and 7 d after infection (between DIV10 and DIV14; blue line). E, Total acid-extracted histone from neurons infected with lentiviruses delivering either shH2afz or shH2afv or both for 6–7 d, resolved by electrophoresis, and blotted for indicated histone. N = 3. Note the cumulative effect of depleting both hypervariants when probed with total H2A.Z antibody. F, IP of nucleosomal H2A.Z from nuclear extract of neurons infected with lentiviruses delivering either shH2afz or shH2afv or both for 6 d resolved by electrophoresis. N = 3. G, H, Neurons were coinfected with indicated constructs for 4–5 d and nuclear extracts were resolved by electrophoresis. IS, insensitive; shRNA target regions were swapped in these constructs. N = 3.