GSM3356370: M. tuberculosis H37Rv, replicate 2; Mycobacterium tuberculosis; RNA-Seq
Total mRNA was extracted with 300 μl of chloroform : isoamyl alcohol (24:1) and precipitated at −20°C overnight with isopropyl alcohol prior to washing with 70% EtOH, then dried and resuspended in 40 μl DEPC‐treated water. RQ1 RNase‐Free DNase (Promega) and TURBO DNase (Ambion) were used to remove contaminating DNA, as recommended by the manufacturers. DNase treatment was followed by RNA purification using one volume of phenol : chloroform : isoamyl‐alcohol (25:24:1) and precipitation using 0.1 volume of 3 M sodium acetate (pH = 5.2) and 0.7 volume of isopropanol. RNA was resuspended in DEPC‐treated water and stored at −80°C. Integrity of RNA was checked by agarose gel electrophoresis, the amount and purity of RNA were assessed using a Nanodrop instrument. RNA-seq libraries were prepared from 1 g of total RNA. The RNA samples were depleted of r-RNAs with the Illumina Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria) then used to generate sequencing libraries with the Illumina TruSeq Stranded mRNA reagents, omitting the polyA selection step (Illumina, San Diego, California, USA). Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on the Illumina HiSeq 2500 using TruSeq SBS Kit v4 reagents.