RNA bound to the XauSPARDA from Xanthobacter autotrophicus Py2
To obtain XauSPARDA-bound nucleic acids, E. coli DH10B was transformed with pBAD_His_Strep_His_XauSPARDA and pCDFDuet_Kn plasmids. Cells were grown at 37 C in LB medium in the presence of 50g/ml ampicillin, 25 g/ml streptomycin and 25 g/ml kanamycin 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). The XauSPARDA-NA complex was purified using a StrepTactin column, all buffer solutions contained 100 mM NaCl. XauSPARDA-bound RNA was purified using phenol-chloroform isolation as described in Zaremba et al., 2022 (doi: https://doi.org/10.1038/s41564-022-01239-0). Then RNA sample was converted to a DNA library using Small RNA-Seq Library Prep Kit (Lexogen cat#052). The resulting DNA library was sequenced using Illumina MiniSeq sequencing with single-end reads and 75 bp read length.