GSM6836430: 11_SMM_Rep2_T5; Streptomyces coelicolor A3(2); RNA-Seq
10 ml of bacterial cultures were 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 neutral pH phenol:chloroform:isoamyl alcohol (25:24:1, Sigma #77617) was added, 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