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: SAMEA110321591
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: ERS12419199
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11935:Eco_RP5_P2_13305_ATCACG
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”. Libraries were prepared using the NEBNext® Small RNA Library Prep Set for Illumina® kit (ref. E7330) according to the manufacturer's protocol. Briefly, 50ng of RNA were subjected to adaptor ligation at 3´and 5´ and first strand cDNA synthesis. After that, PCR selectively enriched those DNA fragments that had adapter molecules on both ends. Library amplification was performed by PCR using NEBNext® Multiplex Oligos for Illumina (Index Primers Set 1, ref. E7335). All purification steps were performed using AgenCourt AMPure XP beads (ref. A63882, Beckman Coulter). Final libraries were analyzed using Agilent Bioanalyzer (ref. 5067-4626) to estimate the quantity and check size distribution. Libraries were quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina's cBot.
characteristics.characteristic: description
characteristics.text: ribosome footprints
characteristics.characteristic: fraction
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Escherichia coli K-12
characteristics.characteristic: organism
characteristics.text: Escherichia coli K-12
characteristics.characteristic: organism
characteristics.text: Escherichia coli K-12
characteristics.characteristic: scientific_name
characteristics.text: Eco_RP5_P2_13305_ATCACG
characteristics.characteristic: title