GSM3262922: Pyruvate at 24 hours, Replicate 1; Mycobacterium tuberculosis; RNA-Seq
The cells were harvested by centrifugation 3,000g at 4°C for 5 min, supernatant was removed and the pellet frozen until the extraction.Total RNA was extracted according to the FastRNA Pro Blue kit manual (MP). The cells were ribolyzed three times (40s /6.0), incubating in ice for 1 minute between pulses. To remove genomic DNA the samples were treated twice with TurboDNase (Ambion) for 1hr at 37°C. Total RNA was purified and concentrated to 20µl of DEPC water using RNA clean-up kit (Zymoresearch) and quantified by Qubit. 2.5ng of RNA were used to check the removal of DNA by PCR . The nucleic acid integrity was verified by Bioanalyzer (Agilent RNA 6000 Nano). RNAs were extracted by FAST Blue kit (MPBio) and suspended in a final volume of 100 ul. The concentration was evaluated by spectrophotometer (Nanodrop, ND-1000) and integrity by RNA6000 Nano reagents and Agilent 2100 Bioanalyzer. 5µg or each RNA samples were depleted of rRNA using the Ribo-Zero Magnetic Kit (Epicentre). The samples were purified using Qiagen RNeasy MinElute Cleanup Kit by means of modified protocol suggested in the Ribo-Zero Magnetic Kit to allow recovering small RNAs. RNA was quantified by Qubit. Libraries were prepared by ScriptSeq v2 (Epicentre cat. SSV21124), FailSafe Enzyme Mix Only (Epicentre) and Index PCR primers (Epicentre, Cat. No. RSBC10948). The libraries was sequenced with an Illumina Hi-Seq 4000 instrument.
GEO Accession
GSM3262922