GSM7389374: H0, sRNA, replicate 1; Mycolicibacterium smegmatis; ncRNA-Seq
Total RNA was isolated by phenol-chloroform extraction and cell disruption using Bead Beater (BioSpec Products, Bartlesville, OK, USA) as described previously (Rustad et al., 2009) and treated with Turbo DNase (Life Technologies, Carlsbad, CA, USA) to remove traces of genomic DNA. To isolate the short RNA fraction the extracted total RNA was subjected to denaturing electrophoresis to separate the size-specific RNA fraction (~ 50-500 nucleotides).The eluted RNA was purified using phenol-chloroform extraction. Total RNA samples were depleted of rRNA using The NEBNext® rRNA Depletion Kit (Bacteria) (NEB, USA) and used to generate sequencing libraries with the NEBNext Ultra II Directional RNA Library Prep Kit (NEB, USA) according to the manufacturers' protocol. The short RNA fraction was sequenced as one replicate per each time point, with no RNA depletion step performed, and the fragmentation step was skipped in order to preserve the integrity of the transcripts.