Illumina MiSeq paired end sequencing; RNA-sequencing of Clostridioides difficile WT and luxS mutant both during mono-culture biofilms and co-culture biofilms with Bacteroides fragilis
RNA-sequencing of Clostridioides difficile WT and luxS mutant both during mono-culture biofilms and co-culture biofilms with Bacteroides fragilis
For biofilm extractions, the biofilms were grown in 24-well tissue culture plates. At the desired time-point, wells were washed once with PBS before adherent cells were disrupted and re-suspended in 1 ml Trizol (Ambion,USA).  The re-suspended bacteria were transferred to lysing matrix tubes (MP Biomedicals, USA) containing 0.1 mm silica beads. Tubes were processed in a FastPrep-24 instrument (MP biosciences) for 6 x 20 second cycles at 6.5 m/s. Following disruption, samples were  centrifuged. Approximately 700 μl of liquid was transferred to fresh RNase-free tubes and incubated at RT for 5 min, before 300 μl chloroform was added. Samples were vortexed for 10 sec and centrifuged at 16,000 x g for 15 min at 4°C. The aqueous phase was transferred to a fresh RNase-free tube containing 200 μl 95% ice-cold ethanol and incubated on ice for 10 min. The mixture was transferred to a spin column assembly (SV Total RNA isolation system; Promega, USA) and centrifuged at 16,000 x g for 1 min. Columns were washed twice with 600 μl and 250 μl RNA washing solution. RNA was eluted in 45 μl RNase free water. DNase treatment was carried out using Turbo DNase kit (Ambion, USA) according to the manufacturer's instruction for rigorous clean-up. RNA was cleaned up using RNeasy mini kit (Qiagen, Germany). The volume of each sample was adjusted to 100 μl with RNase free water and 350 μl of RLT buffer was added. RNA was diluted with 250 μl 100% ethanol. Samples were transferred to RNeasy Mini Spin column and centrifuged at 8000 x g for 15 sec. The flow-through was discarded. Samples were washed twice with 500 μl RPE buffer at 8000 x g for 15 seconds and 2 minutes respectively. Samples were eluted in 45 μl RNase-free water. Following RNA extraction and clean-up, 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. RiboZERO rRNA depletion resulted in 7 μl rRNA-depleted RNA. The Agilent 2100 Bioanalyzer (Agilent, UK) was used to assess both the quality and to confirm rRNA depletion as described above. cDNA libraries were prepared using 5 μl rRNA depleted RNA following the manufacturer's instruction (TruSEQ LT, illumina)
Experimental Factor: organism
Bacteroides fragilis
Experimental Factor: genotype
wild type genotype
Experimental Factor: growth condition
monoculture