title: RNA Seq of Streptomyces albidoflavus: BGC removal/heterologous expression DEL8repl3
design: Total RNA was isolated and purified from liquid cultures of Streptomyces albidoflavus using RNeasy Mini QIAcube Kit. RNA sequencing library preparation was prepared using NEBNext rRNA Depletion Kit (bacteria) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturers instructions (NEB, Ipswich, MA, USA). Briefly, rRNA was depleted with NEBNext rRNA Depletion Kit (bacteria). rRNA depleted RNAs were fragmented. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3ends, and indexed adapter was ligated to cDNA fragments.  Limited cycle PCR was used for library amplification. The dUTP incorporated into the cDNA of the second strand enabled its specific degradation to maintain strand specificity. Sequencing libraries were validated using DNA Kit on the Agilent 5600 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA).