title: PF15 sRNA 37C plate Rep1
design: Bacterial lawns (plate) for RNA collection were cultured as previously described and grown for 2 days on plates at 25C (or 37C for sequencing). 37C liquid cultures for sequencing were pelleted for collection. Bacterial lawns were collected from the surface of NGM plates using a cell scraper. In brief, 1 ml of M9 buffer was applied to the surface of the bacterial lawn, and the bacterial suspension following scraping was transferred to a 15-ml conical tube. PF15 from 10 plates or OP50 from 15 plates was pooled in each tube and pelleted at 5,000g for 10 min at 4C. The supernatant was discarded, and the pellet was resuspended in 1 ml of Trizol LS for every 100 l of bacterial pellet recovered. The pellet was resuspended by vortexing and subsequently frozen at 80C until RNA isolation. To isolate RNA from bacterial pellets, Trizol lysates were incubated at 65C for 10 min with occasional vortexing. Debris was pelleted at 7,000g for 5 min at 4C. The supernatant was transferred to new tubes containing 1/5 the volume of chloroform. Samples were mixed thoroughly by inverting and centrifuged at 12,000g for 10 min at 4C. The aqueous phase was used at input for RNA purification using the mirVana miRNA isolation kit according to the manufacturers instructions for small RNA (<200 nt) isolation. Purified RNA was used immediately for sRNA training, or for sequencing frozen at 80C until further use.-Sample treated with RNA 5' pyrophosphohydrolase (RppH, NEB) at 37 deg C 30 min-converted to Illumina sequencing libraries using the PrepX RNA-seq library preparation protocol on the automated Apollo 324 NGS Library Prep System (Takara Bio)- libraries were examined on Bioanalyzer DNA High Sensitivity chips (Agilent) for size distribution, quantified by Qubit fluorometer (Invitrogen), and then pooled at equal molar amount and sequenced on Illumina NovaSeq 6000 S Prime flowcell