characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-06-29T00:17:05Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-06-29T00:17:05Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110321364
characteristics.characteristic: External Id
characteristics.text: Centre for Genomic Regulation (CRG), the Barcelona Institute of Science and Technology
characteristics.characteristic: INSDC center name
characteristics.text: 2023-06-29T00:17:05Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-06-29T00:17:05Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12418972
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11930:rep2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: Protocols: 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.
characteristics.characteristic: description
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mycoplasma pneumoniae M129
characteristics.characteristic: organism
characteristics.text: Mycoplasma pneumoniae M129
characteristics.characteristic: organism
characteristics.text: 2
characteristics.characteristic: replicate
characteristics.text: Mycoplasma pneumoniae M129
characteristics.characteristic: scientific_name
characteristics.text: rep2
characteristics.characteristic: title