GSM9712871: WT_rep1_uninfected_input biol rep 1; Escherichia coli; RNA-Seq
Pellet was resuspended in 750 µL TRIzol and incubated for 5 min at room temperature. Next, 150 µL of chloroform was added to each sample, incubated for 2 min at room temperature, and then spun down at 12,000g for 15 min at 4 °C. Next, 360 µL of the aqueous phase was transferred to a clean tube and purified with the RNA Clean & Concentrator-5 kit (Zymo Research). RNA was diluted in NEBuffer 2 and heated at 92 °C for 2 min for fragmentation by alkaline hydrolysis. The samples were then treated with TURBO DNase (Thermo Fisher Scientific), RppH (NEB), and T4 PNK (NEB) to remove DNA and prepare RNA ends for adapter ligation. RNA was purified using the Zymo RNA Clean & Concentrator-5 kit. Adapter ligation, reverse transcription, and indexing PCR were performed using the NEBNext Small RNA Library Prep kit. Libraries were sequenced on an Element AVITI system in paired-end mode with 150 cycles per end.