GSM2498342: Δhns_rep2; Mycobacterium tuberculosis H37Rv; RNA-Seq
Exponential phase M. tuberculosis cultures were harvested by centrifugation, pellets were resuspended in TRIzol Reagent (ThermoFisher) and stored at -80° until further processing. Total RNA was extracted by bead-beating as previously described (32). Integrity of RNA was checked by agarose gel electrophoresis, the purity and amount of RNA were assessed using a Nanodrop instrument and Qubit Fluorometric Quantitation (ThermoFisher) respectively. SuperScript III First-Strand Synthesis System (Invitrogen) was used to generate randomly primed cDNA from 500 ng of RNA, according to the manufacturer's recommendations. 300 ng of total RNA were mixed with 5 x Fragmentation buffer (Applied Biosystems), incubated for 4 minutes at 70°C and then transferred immediately to 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 RNeasy MinElute columns (Qiagen). 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 resulting library was purified with AMPure beads (Beckman Coulter) as per the manufacturer's instructions.