GSM1826413
GSM1826413: STR-resubmission rep2; Mycobacterium tuberculosis; RNA-Seq
Forty milliliters of replicating (i.e. with STR) or STR-starved M. tuberculosis 18b cultures were pelleted and cells were flash frozen in liquid nitrogen and stored at -80°C until use. Bacteria were re-suspended in 1 ml Trizol (Invitrogen) and added to a 2-ml screw-cap tube containing 0.5 ml zirconia beads (BioSpec Products). Cells were disrupted by bead-beating twice for 1 minute with a 2-minute interval on ice. The cell suspension was then transferred to a new tube, where chloroform-isoamylalcohol (24:1) extraction was performed. RNA was precipitated by adding 1/10 volume of sodium acetate (2 M, pH 5.2) and 0.7 volume of isopropanol, washed with 70% ethanol, air-dried and resuspended in DEPC-treated water. DNase treatment was carried out twice using RQ1 RNase-free DNase (Promega), following the manufacturer’s recommendations, and the reactions were subsequently cleaned up by phenol-chloroform extraction and ethanol precipitation. RNA was stored at -80°C in DEPC-treated water. Amount and purity of RNA were determined spectrophotometrically and by Qubit analysis (Life Technologies), integrity of RNA was assessed on 1% agarose gel and by Fragment Analyzer (Advanced Analytical). 100 ng of total RNA were used for library preparation according to the instructions provided in the TruSeq Stranded mRNA LT kit (Illumina). An aliquot of the library was analyzed on Qubit (Life Technologies) and Agilent Fragment Analyzer (Advanced Analytical) prior to sequencing on Illumina HiSeq 2000 using the TruSeq SR Cluster Generation Kit v3 and TruSeq SBS Kit v3.
GEO
GSM1826413
GEO Accession
GSM1826413