Illumina HiSeq 2500 paired end sequencing; RNA-seq of Mycoplasma pneumoniae grown in serum-free medium
RNA-seq of Mycoplasma pneumoniae grown in serum-free medium
RNA samples were obtained as follows. Mycoplasma pneumoniae wild-type strain M129 was grown per duplicate in tissue culture flasks containing 5 ml of serum-free medium vB13. After 72h of culture at 37°C under 5% CO2, the culture medium was changed with fresh one, and the cells further incubated for 6h prior RNA extraction. 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 adapter 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.