alias: GSM3377912
title: GSM3377912: M. tuberculosis H37Rv, delta mycP1-eccE1, complemented with eccE1-HA, replicate 2; Mycobacterium tuberculosis; RNA-Seq
library_construction_protocol: 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. Total RNA concentration was measured using the Qubit RNA HS Assay Kit. Libraries of ribosomal-depleted RNA were prepared with 300ng of fragmented RNA using the Ribo-zero rRNA removal kit for Gram positive bacteria and the Truseq Stranded mRNA Library Prep kit reagents (Illumina). RNA-seq libraries were prepared from 1 ug 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.
identifiers.namespace: GEO
identifiers.id: GSM3377912