title: Illumina MiSeq paired end sequencing; RNA-sequencing of WT and luxS mutant Clostridioides difficile during planktonic culture
design: RNA-sequencing of WT and luxS mutant Clostridioides difficile during planktonic culture
library_construction_protocol: Biological triplicates of C. difficile R20291 WT and luxS mutant (insertional ClosTron mutant as described in DOI: 10.1128/JB.01980-12) were grown in planktonic conditions to mid-log phase (approx. 8 hrs) in BHI.  1-2 ml of bacterial culture was pelleted by centrifuge. The supernatant was removed and the cells were re-suspended in 1 ml Trizol (Ambion, USA). Cells were lysed using Fast-prep 24 instrument (MP Bioscience) and RNA was extracted using phenol chloroform. rRNA was depleted from a total of 5 μg of extracted RNA using RiboZERO TM (illumina, UK) according to the manufacturer's protocol, purifying the RNA with ethanol precipitation for optimal RNA recovery. cDNA libraries were prepared using 5 μl rRNA depleted RNA following the manufacturer's instruction (TruSEQ LT, illumina).
attributes.tag: Experimental Factor: genotype
attributes.value: luxS insertional knockout