characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-07-01T00:20:10Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-07-01T00:20:10Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110321593
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: 2023-07-01T00:20:10Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-07-01T00:20:10Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12419201
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11935:MPN_RP10_S1_Tet_15114_CTTGTA
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) for 72h, split into biological duplicates and grown for 6h (exponential phase, standard conditions). In order to block ribosomes at the initiation stage, cells were treated with 40μg/ml of tetracycline during 1 min before lysis. Except for tetracycline-treated samples, cells were treated during 1 min with chloramphenicol at 100 μg/ml final concentration in order to stop translation. After treatment cell media was removed and cells were harvested by scraping and centrifuged at 8000g during 5 min in a 2 ml tube. Supernatant was discarded and cell pellet was resuspended in 1.5ml of lysis buffer, once pellet was resuspended lysis pressure was applied using a prechilled 4639 Cell Disruption Vessel (two passes). The cell lysate was centrifuged in a cooled desktop centrifuge at 16000g for 13 min and the supernatant was frozen into liquid nitrogen for further procedures. In order to block ribosomes at the initiation stage, cells were treated with 40μg/ml of tetracycline during 1 min before lysis. Except for tetracycline-treated samples, cells were treated during 1 min with chloramphenicol at 100 μg/ml final concentration in order to stop translation. In order to get ribosome footprints, a controlled digestion with MNase was performed as described in (Becker et al., 2013). Digestion reaction was stopped with EGTA. With the purpose of isolating ribosomes, a sucrose cushion was performed. We loaded the supernatant lysate onto 45 ml of 25% (for E. coli cells) or 15% (for Mpn cells) sucrose cushion buffer and ultracentrifuged the samples at 45000 rpm during 4h and 30 min at 4 C in a TI-45 rotor Beckman. The cushion buffer was removed carefully with a vacuum system and the pellet resuspended with 200μl of wash buffer. Finally 900μl of Trizol was added in order to perform RNA extraction (manufacture instructions). Once the RNA was purified, we isolated the footprints following the steps described in (Becker et al., 2013) in supplementary methods, page 6, sections “Size selection of the footprint fragments” and “Dephosphorylation”. The RNA-seq libraries were prepared at the CRG Genomics Unit using the TruSeq stranded mRNA Library Prep kit (96 samples ref. 20020595 or 48 samples ref. 20020594) according to the manufacturer's protocol, without the initial polyA selection, to convert total RNA into a library of template molecules of known strand origin and suitable for subsequent cluster generation and DNA sequencing. Briefly, 100 ng of total RNA were fragmented under elevated temperature and primed with random hexamers for cDNA synthesis. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and random primers. After that, second strand cDNA was synthesized, removing the RNA template and synthesizing a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA using DNA Polymerase I and RNase H. To these cDNA fragments, a single 'A' base was added to the 3’ends to prevent self-ligation during the adapter addition. A corresponding single T nucleotide on the 3’ end of the adapter provides a complementary overhang for ligating the adapter to the fragments. Subsequent ligation of the multiple indexing adapter to the ends of the ds cDNA was done. Finally, PCR selectively enriched those DNA fragments that had adapter molecules on both ends. The PCR was performed with a PCR Primer Cocktail that anneals to the ends of the adapters. Final libraries were analyzed using Bioanalyzer DNA 1000 or Fragment Analyzer Standard Sensitivity (ref: 5067-1504 or ref: DNF-473, Agilent) to estimate the quantity and validate the size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit KK4835 (REF. 07960204001, Roche) prior to the amplification with Illumina’s cBot.
characteristics.characteristic: description
characteristics.text: total RNA
characteristics.characteristic: fraction
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: Mycoplasma pneumoniae M129
characteristics.characteristic: scientific_name
characteristics.text: MPN_RP10_S1_Tet_15114_CTTGTA
characteristics.characteristic: title