GSM7058319: Streptomyces coelicolor A3(2) grown In SMM medium, cold-shock, 60 min, replicate 2; Streptomyces coelicolor A3(2); RNA-Seq
10 ml of bacterial cultures were centrifuged and the pellet was resuspended in 200 µl of lysozyme dissolved in TE buffer at a concentration of 0.015 g per ml and incubated at room temperature for 15 min. 600 µl of RNeasy Lysis Buffer (RLT buffer, Qiagen #79216) containing 10 µl/ml b-mercaptoethanol was added and the mixture mixed by vortex mixer then transferred to a 2ml microfuge tube containing 5 mm stainless steel beads (Qiagen 69989) and placed in a Tissuelyser LT (Qiagen #85600) for efficient mechanical cell disruption / homogenisation for 2 min at 30 rpm. The suspension was then centrifuged at 16,627 RCF for 2 min, the supernatant was recovered and its volume estimated. One volume of acid pH 4.5 phenol:chloroform:isoamyl alcohol (125:24:1, Fisher #AM9720) was added (for the first replicate of the solid media cultures neutral pH phenol:chloroform:isoamyl alcohol(25:24:1, Sigma #77617 was substituted)), and the mixture mixed by vortex mixer for 30 seconds, then centrifuged at 4°C for 5 min at 16,627 RCF. The top phase containing the nucleic acids was removed and retained leaving the interface, and the phenol/chloroform extraction repeated twice more. Then one volume of chloroform was added, the mixture mixed by vortex mixer then centrifuged at 4°C for 2 min at 16,627 RCF, and the top phase was again transferred to a fresh RNase-free microfuge tube. From this point onward the standard manufacturer's instructions were followed for the miRNAeasy RNA extraction kit (Qiagen #217004). The total RNA was then eluted in 30 µl of nuclease free water and stored at -80°C. Genomic DNA was removed using the Turbo DNA free kit (Invitrogen AM1907). NEBNext® Ultra™II Directional  RNA Library Prep Kit for Illumina® (cat. E7760) according to manufacturer's instructions. rRNA depletion was performed using NEBNext rRNA Depletion Kit (Bacterial) (NEB E7850) for the rich liquid media samples, the Illumina Ribo-zero rRNA Removal Kit (Bacteria)(Illumina MRZMB12424) for the minimal solid media samples and using the NEBNext RNA Depletion Core Reagent Set (NEB E7865) with custom DNA probes for the minimal liquid samples.