GSM9111116: P. aeruginosa, planktonic growth, treated with midazolam 10 ug/ml, replica 3; Pseudomonas aeruginosa; RNA-Seq
RNA extraction from planktonic cells. Briefly, 10 mL of LB containing the different treatments (untreated control or treatment with Midazolam 10 µg/ml) were inoculated with 500 μL of an overnight PAO1 culture and grown until OD600nm0.5-0.6 at 37ºC and 180 rpm. One millilitre of the culture was mixed with 2 ml of RNA protect (QIAgen) and the RNA extraction was performed using the RNeasy kit (QIAgen). Contaminant DNA was removed using 2 U of the TURBO DNAse kit (Invitrogen) and incubated for 30 minutes at 37ºC. Bacterial RNA was extracted from three independent replicates for each experimental condition. RNA extraction from biofilms. Biofilms were prepared as previously explained except adding 1 mL of the normalised culture into a 24-well plate (untreated control or treatment with Midazolam 10 µg/ml). After 24h at 37ºC, RPMI media was replaced by 1 mL of 0.9% NaCl and biofilm was thoroughly scratched from the well and transferred to a 1.5 mL tube. A bath sonicator was used to disaggregate the biofilm embedded cells prior RNA extraction. RNA extraction was performed as previously explained but adding an extra DNA degradation step (RNase-Free DNase, QIAgen). The TURBO DNAse was also applied after RNA elution, doubling the enzyme amount used and increasing the incubation time up to 45 minutes. RNA was prepared from three independent biofilm cultures. Library Construction, Quality Control and Sequencing. Firstly, ribosomal RNA was removed from total RNA using the Illumina Ribo-Zero Plus rRNA Depletion Kit followed by ethanol precipitation. For the library preparation, the Novogene NGS Stranded RNA Library Prep Set (PT044) was used.After fragmentation, the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and 2 sequenced on an Illumina NovaSeq 6000 using the PE150 strategy, according to effective library concentration and data amount required. Data were unequally shared with 3G raw data (10 million paired reads) for each sample.