GSM5770592: M9 Min Glu #1, RNA, replicate 2; Escherichia coli; RNA-Seq
The pellet was resuspended in 100 μl of TE and treated with 177kU (1 μl) Ready-lyse lysozyme solution (Lucigen: Catalog #R1804) and 0.2 mg (10 μl) proteinase K (Thermo Fisher Scientific: Catalog #EO0492), incubated for 10 min at room temperature with vortexing every 2 min. The RNA was purified using RNA Clean and Concentrator kit-5 (Zymo: Catalog #R1014), treated with 5 units of Baseline-ZERO DNase (Epicentre: Catalog #DB0715K) in the presence of RNAse inhibitor (NEB: Catalog #M0314L) for 30 min at 37°C. RNA was purified again using RNA Clean and Concentrator kit-5 (Zymo: Catalog #R1014). RNA was flash-frozen in a dry ice-ethanol bath and stored at −80°C. rRNA depletion was performed using the bacterial rRNA depletion kit following manufacturer instructions (New England Biolabs (NEB): Catalog #E7850L). The only modification performed was the last step, where instead of a bead clean up, we used the RNA Clean and Concentrator kit-5 (Zymo: Catalog #R1014).  Sequencing preparation was performed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina following manufacturer instructions (NEB: Catalog #E7420L) for rRNA-depleted RNA.  Random primers were used, and RNA was considered intact for the NEBNext Ultra protocol. Slight modifications to the protocol are as follows. To purify cDNA, the Oligo clean & concentrator was used (Zymo: Catalog #D4061). Following adapter ligation, DNA was puri- fied using DNA Clean & Concentrator-5 (Zymo: Catalog #D4014).