Extended Data Fig. 6: Validation of SPT2 antibodies and siRNAs, and controls for H3.3-SNAP experiments.

a, Western blot against HsSPT2 in total extracts of SPT2-depleted U-2 OS cells; one representative experiment of three. b, Expression of GFP-tagged HsSPT2, full-length or mutants, in U-2 OS Flp-In cells; one representative experiment of two. c, Soluble/Chromatin fractionation of U-2 OS Flp-In cells expressing GFP-tagged HsSPT2, full-length or mutants; one representative experiment of two. d, e, H3.3-SNAP U-2 OS cells were treated with the indicated siRNAs, and newly synthetized H3.3-SNAP was labelled 48 hours post-transfection. New TMR-labelled H3.3-SNAP fluorescence intensity after (d) pre-extraction or (e) direct fixation is shown. n = 3, data represented as mean ± S.D. Statistics: one-way ANOVA with Dunnett’s post-test. ns: P > 0.05, *: P < 0.05, ***: P < 0.0001. One-way ANOVA results, Pre-extraction: P = 0.0048, Direct fixation: P = 0.3447. Supplementary Table 3: P values of Dunnett’s post-tests. f, Total extracts of siRNA-treated U-2 OS cells, 48 hours post-transfection. One representative experiment of two. g, Soluble/Chromatin fractionation of siRNA-treated U-2 OS cells, 48 hours post-transfection. One representative experiment of two. h, qPCR for H3.3-SNAP expression in cells harvested 48 hours post-transfection. n = 3, data represented as mean ± S.D. Statistics: one-way ANOVA with Dunnett’s post-test. One-way ANOVA results, P = 0.4767. Supplementary Table 3: P values of Dunnett’s post-tests. i, SPT2 levels in the indicated H3.3-SNAP U-2 OS cell lines; one representative experiment of three. j, k, New TMR-labelled SNAP-H3.3 fluorescence intensity in the indicated U-2 OS SNAP-H3.3 cell lines after (j) direct fixation or (k) pre-extraction. Red line: median. One representative experiment of four, medians from all experiments plotted in Fig. 6e, f. Direct fixation, WT: n = 2487, KO-3: n = 3891, KO-3 + SPT2^WT: n = 572, KO-3 + SPT2^HBM: n = 1278, KO-4: n = 4693, KO-4 + SPT2^WT: n = 1302, KO-4 + SPT2^HBM: n = 924; Pre-extraction, WT: n = 4617, KO-3: n = 6673, KO-3 + SPT2^WT: n = 1382, KO-3 + SPT2^HBM: n = 1864, KO-4: n = 6725, KO-4 + SPT2^WT: n = 2101, KO-4 + SPT2^HBM: n = 1472. l, qPCR for H3.3-SNAP expression. n = 4, data represented as mean ± S.D. Statistics: one-way ANOVA with Dunnett’s post-test, calculated independently on SPT2 KO-3 or KO-4 cells. One-way ANOVA results: KO-3: P = 0.3754, KO-4: P = 0.7420. Supplementary Table 3: P values of Dunnett’s post-tests. m, ATAC-seq levels at a control subset of 100 random, non-differentially accessible genes.

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