Extended Data Fig. 3: ASO knockdown specifically downregulates targets.
From: Programmable antisense oligomers for phage functional genomics
PAO1 cells were pretreated with 6 µM ASO against chmA, ΦKZ055 (nvRNAP), and picA for 30 min. Cells were infected with ΦKZ at an MOI = 5 and incubated for 2.5, 5.0, 7.5, and 10 min followed by harvesting in non-reducing SDS-PAGE loading dye. Subsequently, proteomics was performed on the samples and proteins were quantified by label-free quantification (LFQ). a. Counts for host proteins at all timepoints were pooled to calculate enrichment and log10 p-value (calculated with MaxQuant Perseus, a two-sided Student’s t-test was used with no adjustments for multiple comparisons). Filter criteria were applied (only one protein in protein group, >=4 unique peptides, >40 peptide posterior error probabilities score, sum of average LFQ intensity was filtered at >1E8 counts), n = 1,130 host proteins were considered. Host protein levels were not altered in the range log2 fold change (log2FC) <-2 or >2 and a –log10 p-value > 2. b. Counts for phage proteins at 7.5 and 10 min p.i. timepoints were pooled to calculate enrichment and log10 p-value (calculated with MaxQuant Perseus, a two-sided Student’s t-test was used with no adjustments for multiple comparisons). Filter criteria were applied (sum of log2LFQ counts for all three ASO treatments >25 counts); n = 95 phage proteins were considered. Log2FC was calculated based on average counts. When a protein was not detected, we set a pseudo-count=1. ΦKZ016 and ΦKZ165 were lower in the non-targeting ASO control sample and therefore omitted. The structural protein ΦKZ094 was only detected in the sample treated with an ASO targeting chmA at 7.5 min p.i. but not at 10 min or in the control sample and was also omitted. c-e. Left, Schematic overview of the transcriptional units (TU) of the targeted transcripts, based on Putzeys et al. 2024 (ref. 49). The position of ASO is indicated. Right, Log2FC of protein levels at 10 min p.i. based on LFQ counts. Source data for panel (a) and (b) are available online.
