title: Sample 6
organism: Streptococcus pyogenes
description: Protocols: Bacteria grown to OD= 0.8 were quickly centrifuged and directly used or frozen in a dry ice/ethanol bath and stored at -80 °C. Bacteria were cultured to late-exponential phase (OD 0.8) in Todd-Hewitt broth with yeast extract (THY) supplemented or not with 15 mM MgCl2. For each condition triplicate cultures were obtained Bacterial pellets were resuspended in 400 µl 10% glucose, 60 mM EDTA, 12.5 mM Tris pH 7.6. Bacteria cell walls were broken by using two cycles in FastPrep (FastPrep FP120 homogenizer- speed: 6, time:30 s) at 4 \C, separated by 1 min., in the presence of 0.4 g of 200-300 µM glass beads and 500 µl acid phenol pH 4.5. After centrifugation the aquaeous phase was extracted once with 1 ml trizol and 100µl phenol:chloroform:isoamyl alcohol (25:24:1 volume ratio) and once with 200 μl chloroform:isoamyl alcohol and RNA was precipitated by isopropanol.Traces of DNA were removed by using the Turbo DNA-free kit(Life Technologies) according to manufacturer's instructionq. RNA quality was controled on Agilent 2100 Bioanalyzer with RNA 6000 nanochip and only RNA with RNA Integrity Number (RIN) higher than 9 was used.8 µg total RNA from each RNA sample was depleted in rRNA by using the MICROBExpress bacterial mRNA enrichment kit and rRNA depletion was controled on Agilent Bioanalyzer. For each of the strand-specific RNA-seq library preparation, depleted RNA corresponding to 1 µg of total RNA was used. RNA was treated with TAP (Tebu-Bio), fragmented using the RNA fragmentation Reagent kit(Ambion). Dephosphorylation of the fragmented RNA by using the antarctic phosphatase (Biolabs), and rephosphorylation with the T4 polynucleotide kinase (Biolabs) were followed by a purification step through RNeasy MinElute Cleanup columns (Qiagen). The RNA was then successively ligated to the 3′ RNA adapter (Illumina TruSeq Small RNA kit) by using truncated T4 RNA ligase 2 (Biolabs) and to the 5′ RNA adapter (Illumina TruSeq Small RNA kit) with T4 RNA ligase (Biolabs). RNA ligated to the two adapters was reverse-transcribed with the Superscript II Reverse Transcriptase (Life Technologies) and the RNA reverse transcription primer (Illumina TruSeq Small RNA kit). The resulting cDNAs were amplified for 13 PCR cycles with Phusion Taq polymerase using the RP1 primer and one of the indexed PCR primers (Illumina TruSeq Small RNA kit).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-11-30T11:01:58Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-08-01T09:17:07Z
attributes.tag: External Id
attributes.value: SAMEA4810940
attributes.tag: INSDC center name
attributes.value: IP
attributes.tag: INSDC first public
attributes.value: 2018-11-30T11:01:58Z
attributes.tag: INSDC last update
attributes.value: 2018-08-01T09:17:07Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7101:Sample 6
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: THY broth
attributes.tag: sample name
attributes.value: E-MTAB-7101:Sample 6
attributes.tag: scientific_name
attributes.value: Streptococcus pyogenes
attributes.tag: strain
attributes.value: S119