RIP-seq of RNA associated in vivo with the PNPase and PNPaseD493A proteins, performed in Bacillus subtilis 9407
PNPase is the primary RNA turnover enzyme in Bacillus subtilis and plays a crucial role in regulating gene expression. To investigate the life activities that PNPase participated in Bacillus subtilis 9407, RNA immunoprecipitation-sequencing (RIP-seq) analysis was performed to pinpoint the direct RNA targets of PNPase using  6×his-tagged PNPase  (PNPase group) and PNPaseD493A (D493A group) constructs that were driven by the IPTG-induced Pgrac promoter. Overall design: The wild-type pnpA CDS with a 6×His tag was integrated into the plasmid pHT01, which contains the IPTG-induced Pgrac promoter, to create a pHT01 derivative vector pXF11. Similarly, the pnpA D493A CDS with a 6×His tag containing the active site D493A mutation was also integrated into pHT01 to obtain another pHT01 derivative vector pXF15. Subsequently, the PNPase deletion strain (?pnpA: GXF1006) was transformed with pXF11 and pXF15 by electroporationto obtain strain GXF2015 (expressing active PNPase protein) and GXF2023 (expressing inactive PNPase D493A protein), respectively. GXF2015 and GXF2023 were grown to the exponential growth phase in the presence of 0.1 mM IPTG induction. The 10% lysis sample was stored and labeled as "Input". 80% of the sample was used in immunoprecipitation reactions with anti-6×His antibody (Abcam) and referred to as "IP", while the remaining 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control, named "IgG". RNA Immunoprecipitation (RIP) was conducted using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Sigma-Aldrich) and 6×his monoclonal rabbit antibodies (Abcam), following the manufacturer's instructions. The target RNAs of "Input" and "IP" samples were extracted using TRIzol reagent (Invitrogen). The stranded RNA sequencing library was constructed using KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (Seqhealth) following the manufacturer's instruction. The library products corresponding to 200-500 bps were enriched, quantified, and finally sequenced using the PE150 model on a Novaseq 6000 sequencer (Illumina).