Illumina MiSeq paired end sequencing; The transcriptional regulator LysG of Mycobacterium tuberculosis activates lysE in a lysine-dependent manner
The transcriptional regulator LysG of Mycobacterium tuberculosis activates lysE in a lysine-dependent manner
Harvesting of liquid cultures was performed by centrifugation and freezing in liquid nitrogen. RNA from bacteria was extracted from a growing culture at OD600 of ~0.3. The cells were incubated in an equal amount of 5 M GTC buffer (5 M guanidinium isothiocyanate, 0.5% n-laurylsarcosine, 0.7% sodium citrate, 0.7% ?-mercaptoethanol) for 15 min at room temperature and harvested by centrifugation at 4,500 x g for 15 min. The pellet was resuspended in 1 ml of TRIzol RNA Isolation Reagent (Life Technologies, Darmstadt, Germany) and incubated for further 15 min at room temperature. Cell lysis was done in a Lysing Matrix B tube (MP Biomedicals, Illkirch, France) using the Hybaid RiboLyser. The tubes were centrifuged at 13,000 x g for 3 min and the supernatant was transferred in a new reaction tube for chloroform extraction. The aqueous solution, consisting of the RNA, was mixed with an equal volume of 70% ethanol and the purification was performed with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the protocol given by the manufacturer. In deviation from the protocol, the optional DNase I digestion (DNase I supplied by Qiagen) was extended to 1 h and a second DNase I digestion (DNase I supplied by NEB) was performed after elution from the columns. A second RNA cleanup was done using the RNeasy Mini Kit. RNA quality and quantity was checked by an Agilent 2100 Bioanalyzer run (Agilent Technologies, Böblingen, Germany) and Trinean Xpose sytem (Gentbrugge, Belgium) prior and after rRNA depletion by Ribo-Zero rRNA Removal Kit (Bacteria) (Illumina, San Diego, CA, USA). TruSeq Stranded mRNA Library Prep Kit from (Illumina,  (San Diego, CA, USA) was used to prepare the cDNA libraries to analyze the whole transcriptome. additional library was prepared from a pool of all isolated RNA according to the protocol of Pfeifer-Sancar et al. PMID:     24341750  focusing on native 5'-ends of transcripts.
Experimental Factor: genotype
del_ysG (1985c)
Experimental Factor: growth condition
10 millimolar ammonium as sole nitrogen source