GSM9177700: Expression of MFP5, intact rhaBADM, replicate 1; Escherichia coli; RNA-Seq
Total RNA was extracted from cultures following the protocol in the RNAprotect Bacteria Reagent Handbook (Qiagen). Once main cultures reached OD600 = 2, 2.5 mL culture was added to 5 mL RNAprotect Bacteria Reagent (Qiagen), vortexed, and incubated at room temperature for 5 minutes. Sample pellets were resuspended in 400 L elution buffer and split into two aliquots which were stored at -80C. Total RNA was then extracted and purified from thawed pellets on a QIAcube Connect Instrument (Qiagen) using the RNeasy Protect Bacteria Mini Kit (Qiagen). Lysozyme was used for enzymatic lysis of cells together with Proteinase K for degrading protein and SUPERaseIn RNAse inhibitor (Thermo Fisher) for protecting RNA. A DNase treatment step was added to the protocol to remove DNA contamination. Total RNA was eluted in 30 L RNase-free water, then stored at -80 C. The extracted RNA was shipped to sequencing at Azenta Life Sciences. Enrichment, cDNA synthesis, and adapter ligation