GSM1310817: WT [RNA-Seq]; Mycobacterium tuberculosis H37Rv; RNA-Seq
Bacteria were resuspended in 1 ml RNA Protect Bacteria Reagent (Qiagen), incubated for 5 min at room temperature and then centrifuged. Bacterial pellets were resuspended in 0.4 ml lysis buffer (0.5% SDS, 20 mM NaAc, 0.1 mM EDTA) and 1 ml phenol:chloroform (pH=4.5) 1:1. Suspensions were transferred to tubes containing glass beads (Qbiogene) and lysed using a ribolyser (Fast-prep instrument) with a three-cycle program (15 sec at speed 6.5 m) including cooling the samples on ice for 5 min between pulses. Samples were then centrifuged and the homogenate was removed from the beads and transferred to a tube containing chloroform:isoamylalcohol 24:1. Tubes were inverted carefully before centrifugation and the upper (aqueous) phase was then transferred to a fresh tube containing 0.3 M Na-acetate (pH=5.5) and isopropanol. Precipitated nucleic acids were collected by centrifugation. The pellets were rinsed with 70% ethanol and air dried before being re-dissolved in RNase-free water. DNA was removed from RNA samples using Turbo DNA free (Ambion) by incubation at 37°C for 1 h. RNA integrity was assessed by agarose gel electrophoresis and absence of contaminating DNA was checked by lack of amplification products after 30 PCR cycles. 10 μg total RNA were treated with 10 U of Terminal 5’-phosphate dependent Exonuclease (Epicentre) for 24 h at 30°C followed by phenol extraction and isopropanol precipitation. Successful preparation of primary transcriptome was confirmed by lack of 23S/16S rRNA bands and presence of intact 5S rRNA  in agarose gels. 100 ng of total RNA were mixed with 5x Fragmentation buffer (Applied Biosystems), incubated for 4 minutes at 70°C and then transferred immediately on ice. RNA was purified using RNAClean XP beads (Beckman Coulter), according to the manufacturer’s recommendations, and subsequently treated with Antarctic phosphatase (New England Biolabs). RNA was then re-phosphorylated at the 5’-end with polynucleotide kinase (New England Biolabs) and purified with Qiagen RNeasy MinElute columns. In order to ensure strand-specificity, v1.5 sRNA adapters (Illumina) were ligated at the 5’- and 3’-ends using RNA ligase. Reverse transcription was carried out using SuperScript III Reverse Transcriptase (Invitrogen) and SRA RT primer (Illumina). Twelve cycles of PCR amplification using Phusion DNA polymerase were then performed and the library was finally purified with AMPure beads (Beckman Coulter) as per the manufacturer’s instructions. A small aliquot (2.5 ml) was analyzed on Invitrogen Qubit and Agilent Bioanalyzer prior to sequencing on Illumina HiSeq 2000 using the TruSeq SR Cluster Generation Kit v3 and TruSeq SBS Kit v3.
GEO Accession
GSM1310817