Extended Data Fig. 2: The quality control of the pooled library and the screen result.
From: METTL3-based epitranscriptomic editing screening identifies functional m6A sites in cancers
a) m6A peak profiles along SETD7 transcripts from meRIP-Seq in multiple prostate and lung cancer cell lines, targeted peak boxed in red. b-c) Gini index of sgRNAs read evenness (b) and mapping rate (c) in pooled plasmid libraries. d) Spearman correlation of normalized counts in the 22Rv1 screen (T0_rep1-3 and T16 (T16_rep1-3); all pairwise r > 0.8, with stronger clustering observed within the T0 and T16 groups. e-f) Gini index (e) and mapping rate (f) for screen samples at T0 and T16 (3 replicates each). g) CDF plot of guide-level negative scores for sample-targeting (orange) vs non-targeting controls (blue). h) Volcano plot showing differentially expressed genes in 22Rv1. Vertical dashed lines indicate the -log2(1.2) and log2(1.2); the horizontal dashed line marks the P = 0.05. i) Western blot of dCasRx-M3 and endogenous METTL3 in H358, H2122, H1299, and LNCaP. GAPDH as a loading control. Representative of two independent experiments with similar results. j) m6A meRIP-qPCR quantification of SETD7 writing efficiency in four cell lines. Results were mean ± SD; n = three independent experiments. Fold change values of gSETD7 relative to gNT mean value were shown. Statistical significance was evaluated using an unpaired two-sided Student’s t-test. k) CDF of guide-level negative scores in H358 (Sample-targeting, orange; non-targeting, blue). l) Volcano plots of enriched or depleted guides in H358 cells. Vertical dashed lines represent log2 fold change -0.5 and 0.5; the horizontal dashed line marks an FDR threshold of 0.1. m) Volcano plots of differentially expressed genes (average |FC | > log2(1.2), P value < 0.05) in H358 after pooled screening. Vertical dashed lines indicate -log2(1.2) and log2(1.2); the horizontal dashed line marks adjusted P = 0.05.
