Illumina HiSeq 2500 paired end sequencing; RNA-seq of Lon and FtsH conditional mutants of Mycoplasma pneumoniae under depleting and inducing conditions
RNA-seq of Lon and FtsH conditional mutants of Mycoplasma pneumoniae under depleting and inducing conditions
RNA samples were obtained as follows. M. pneumoniae strains ΔIndLon, ΔIndFtsH and ΔIndLon_FtsH were grown in 5 ml cultures supplemented with 20 µg/ml of chloramphenicol and 10 ng/ml of tetracycline to induce Lon and/or FtsH expression. After 48h of culture, cells were washed with Hayflick twice and scraped off from the flasks in 5 ml of fresh medium without tetracycline. To grow cells under inducing or depleting conditions, new 5 ml cultures containing plain medium (depletion) or supplemented with 100 ng/ml tetracycline (induction) were inoculated with 1:5 or 1:12.5 dilutions of cell suspension, respectively. RNA samples were obtained after 48h (to deplete Lon) or 72h (to deplete FtsH) of depletion. Before RNA isolation, the culture medium was changed with fresh one and the cells further incubated for 6h. Cells were washed with PBSx1 and lysed immediately with 700μl Qiazol (Qiagen). RNA was isolated using the miRNeasy kit (Qiagen) following the manufacturer's instructions, including the in-column DNase I treatment. The quality of RNA (amount and integrity) was assessed using a BioAnalyzer (Agilent).  RNA-seq libraries were prepared at the CRG ultrasequencing facility using the TruSeq Stranded mRNA Sample Prep Kit v2 according to the manufacturer's protocol using the following modifications. The poly(A) selection step was omitted and fragmentation was done using 100 ng total RNA as starting material. To maintain smaller library insert sizes than in the standard protocol, the first AMPure XP purification after adaptor ligation was performed using 50 ul AMPure XP beads instead of 42 ul. The second round of bead purification was then performed using 55 ul AMPure XP beads instead of 50 ul. The purification of the PCR reaction after library amplification was done using 55 ul AMPure XP beads instead of 50 ul.