Transcriptomic Sequencing of E. coli W3110 and a W3110 speB mutant
Cells were grown for 2 hours in 1 mL GT media. 60 L were then added to 1 mL of fresh GT, with and without 1 mM putrescine and grown for another two hours. After growth, the cells were centrifuged, and frozen at -80C. Cell pellets were thawed, resuspended in 0.7 mL of buffer RLT and mechanically lysed using a bead beater (FastPrep-24 Classic from MP Biomedical) set at the highest setting for three 45-s cycles with a 5-min rest period on ice between cycles. Cell lysates were used to isolate RNA using a Qiagen Rneasy mini kit. RNA was quantified, DNase treated, and re-quantified on a nanodrop. RNA that passed the first quality check was analyzed on RNA-IQ (Qubit). RNA with an IQ higher than 7.5 was used. RNA that passed both checks were run on an agarose gel. RT-qPCR was used to ensure there was no DNA contamination. Lexogen RiboCop rRNA depletion kit for Bacteria followed by Illumina Truseq Stranded mRNA library prep kit.