GSM9191300: Rep-2_0% Nitrate_5 _DO_biofilms; Mycolicibacterium smegmatis MC2 155; RNA-Seq
RNA extraction was performed using the Qiagen miRNeasy Mini kit (Cat. No. 1038703). The samples were homogenized and lysed in TRIzol reagent using Tissuelyser at 20 Hz for 1 minute, extracted with chloroform and the aqueous phase separated and mixed with 1.5 times (v/v) chilled ethanol (100%). This homogeneous mix was loaded on the column (provided by kit), centrifuged for 8,000 Xg for 60 seconds and the flow-through was drained off. The subsequent steps were followed as instructed on the kit, with an on-column DNase I digestion step to remove contaminating DNA and to elute RNA with 50 μl of RNase free water. 250ng of RNA sample was used for ribodepletion by using NEBNext rRNA depletion kit Bacteria (Cat. no. NEB E7850X), according to the manufacturer's protocol. The NEBNext Ultra II Directional RNA Library prep kit for Illumina (Cat. no. E7760L), was used to construct double-stranded cDNA libraries.