Fig. 2

Blocking primers in EMBR-seq deplete rRNA and provide a deeper view of the transcriptome without introducing technical biases. a In the presence of blocking primers, a 4-fold rRNA depletion and more than 2-fold mRNA enrichment is achieved compared to control samples. With the introduction of blocking primers in EMBR-seq, mRNAs account for more than 80% of the mapped reads, which is a greater than 16-fold increase compared to total RNA in E. coli cells. The 3′ phosphorylated blocking primers display similar but slightly lesser mRNA enrichment (n ≥ 2 replicates for all conditions). b Comparison between EMBR-seq and control samples in the number of genes detected above different expression thresholds (n = 3 for both conditions). For the EMBR-seq group, error bars are of the same scale as the size of the data points. c Venn diagram shows that more than 99% of the genes detected in the control samples were also detected when using blocking primers in EMBR-seq. 99.2% of all detected genes were found in the EMBR-seq samples and 96.2% in the control samples. The number of genes detected were calculated by combining data obtained from three control samples and three EMBR-seq processed samples. d Gene transcript counts with and without blocking primers are highly correlated (Pearson r = 0.97) suggesting that EMBR-seq does not introduce technical artifacts in quantifying gene expression (n = 3 for both datasets). These experiments were performed starting with 100 ng total RNA from E. coli. Error bars in panels (a) and (b) represent standard deviations