Name: rep2_p
Instrument: Illumina HiSeq 2500
Strategy: ncRNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: PAIRED
Construction protocol: M. pneumoniae cell cultures were grown in Haylick rich medium as previously described (Yus et al., 2009). Briefly, M. pneumoniae strains were grown per duplicate in tissue culture flasks containing 5 ml of Hayflick medium supplemented with 20 µg/ml of chloramphenicol. After 48h 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). Eight to ten micrograms of Total RNA were loaded on a 15% denaturing urea polyacrylamide gel (EC6885BOX, Thermo Scientific) and RNA fragments of 60 to 100nt were selected. They were then subjected to limited alkaline hydrolysis, followed by dephosphorylation using Antarctic phosphatase (M0289, New England Biolabs) and rephosphorylation using PNK (M0201, New England Biolabs). RNA-seq libraries were prepared at the CRG ultrasequencing facility. Briefly, 3 prime and 5 prime adapters were ligated to the RNA fragments, and these were subsequently reverse transcribed and amplified by PCR, where different indexed primers were used to allow multiplexed sequencing. Final libraries were analyzed using Agilent High Sensitivity chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina's cBot.