total RNA from E. coli BL21-AI cells with XauSPARDA from Xanthobacter autotrophicusPy2 and pCDFDuet plasmid
To obtain total RNA with XauSPARDA E. coli BL21-AI was transformed with pBADHisA_XauSPARDA and pCDFDuet plasmids. Cells were grown at 37 C in LB medium in the presence of 50g/ml ampicillin and 25 g/ml streptomycin until OD600=0.5 was reached. Then, expression was induced by adding 0.2% w/v L-arabinose, and cells were harvested after 2 h. Cells were disrupted by incubating 20 min at room temperature in lysis reagent B-PER (ThermoFisher cat#78243) containing 3 mg/ml lysozyme (ThermoFisher cat#89833). Total RNA was purified using SPLIT RNA Extraction Kit (Lexogen cat#008). Ribosomal RNA was excluded using RiboCop rRNA Depletion Kit (Lexogen cat#126). Then RNA sample was converted to a DNA library using CORALL Total RNA-Seq Library Prep Kit (Lexogen cat#095). The resulting DNA library was sequenced using Illumina MiSeq sequencing with pair-end reads and 150 bp read length.