GSM1406375: BTP15 rep1; Mycobacterium tuberculosis; RNA-Seq
Bacteria were resuspended in 600 µl of Trizol and lysed in with 400 µl of glass beads in a bead beater. After spinning and recovery of the supernatant, 200 ul of chloroform were added. After shaking and spinning, the supernatant was purified using the RNeasy bench protocol. 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. 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.