Fig. 6: Direct identification of tRNA from E.coli LMW RNA extracts.

a Isolation of LMW RNA from E.coli. (I) E.coli pellets were lysed in the RNAiso buffer (Takara). (II) LMW RNA was extracted with chloroform and retained in the supernatant. (III) After centrifugation, the supernatant was collected and added with isopropanol to precipitate all LMW RNA. (IV) The precipitant was collected and washed with 75% ethanol. (V) The LMW RNA was dissolved in ribonuclease (RNase)-free water. b Denaturing urea polyacrylamide gel electrophoresis (Urea-PAGE) analysis of E.coli LMW RNA, extracted as described in a. L1: low range RNA ladder. L2-L4: E.coli LMW RNA. The band corresponding to tRNA is marked on the gel. The uncropped gel is provided in Supplementary Fig. 28. c A representative trace containing successive translocation of E.coli LMW RNA through MspA. Characteristic tRNA type 1 and type 2 events are respectively marked with blue and red triangles. d Zoomed-in views of representative translocation events in c, which were marked with i and ii on the trace. e The proportion of tRNA translocation events (purple: 5 S rRNA. red: tRNA. gray: others). 48% of all acquired events were recognized as either tRNA type 1 or tRNA type 2 events. Error bars represent standard deviation, n = 3 independent replicates. The measurements in c–e were performed as described in Methods. E.coli LMW RNA was added to cis with a final concentration of 40 ng/μL. Trace segmentation and event recognition were performed with the custom machine learning algorithm (Fig. 3a).