Fig. 2: Validation of smRandom-seq using reference bacteria samples.
From: Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq
a Scatter plot of UMI counts per cell barcode in the mixture. E. coli number: 202. B. subtilis number: 45. Mixed cell number: 4. b Species specificity of UMI in the mixture. E. coli number: 202. B. subtilis number: 45. Data in the box plot in b, c, j corresponded to the first (lower hinges) quartiles, third quartiles (upper hinges), and median (center). c Distribution of detected genes of different bacterial species. The identified cell numbers were as follows: 45 B. subtilis, 5001 E. coli, 1105 A. baumannii, 1127 K. pneumoniae, 2973 P. aeruginosa, and 1989 S. aureus. d Representative proportions of transcript categories of E. coli samples with and without rRNA depletion. The cell number of the E. coli samples in d–f was 7645. e Scatter plot showing detected genes and the number of reads per barcode of E. coli samples with (blue) and without (red) rRNA depletion. f Correlation of gene expression (Log10(Counts + 1)) of E. coli samples with and without rRNA depletion. The E. coli sample with rRNA depletion was sequenced more deeply for the next performance evaluations. g The barcode rank plot of log10(total number of detected genes per barcode) versus log10 (Barcode number) for each possible barcode. The knee point (red dashed line) indicated the threshold for putative E. coli (cell number: 7645). h Saturation curve for the rRNA depleted and deeper sequenced E. coli sample (Cell number: 7645). i Comparison of performance with other two reported high-throughput single bacterium RNA-seq methods (PETRI-seq:15 exponential-phase E. coli dataset from experiment 2.01, microSPLiT16: E. coli dataset form the E. coli and B. subtilis species-mixing experiment). j Comparison of qPCR relative expression (n = 3 biologically independent samples) and UMI count of E. coli samples (E1 n = 252 cells, E2 n = 254 cells, E3 n = 254 cells, E4 n = 253 cells) with a progressive increase in expression of GFP. E1–E4 represents the GFP-E. coli samples induced with 0, 6.25, 25, and 100 mM propionate. k UMAP projection of two repeated E. coli samples (Repeat 1 and Repeat 2) applied with smRandom-seq separately. Source data are provided as a Source Data file.
