alias: GSM3529945
title: GSM3529945: MVU_KOWhiB6_2; Mycobacterium marinum M; RNA-Seq
library_construction_protocol: M. marinum and M. tuberculosis cultures were pelleted and bead beated in 1 ml of TRIzol (Invitrogen) with 0.1-mm zirconia/silica beads (BioSpec Products). After centrifugation, supernatants were extracted with chloroform, and RNA was precipitated with isopropanol. RNA pellets were washed with 80% ethanol and dissolved in RNAse-free water. Contaminant DNA was removed by incubation with DNAse I (Fermentas). Total RNA was extracted with TRIzol (Invitrogen) and then purified on RNeasy spin columns (Qiagen) according to the manufacturer's instructions. RNA integrity (RNA integrity score ≥ 6.8) and quantity were determined on an Agilent 2100 Bioanalyzer (Agilent; Palo Alto, CA, USA). As ribosomal RNA constitutes a vast majority of the extracted RNA population, depletion of these molecules via RiboMinus-based rRNA depletion was conducted. For mRNA enrichment, Invitrogen's RiboMinus Transcriptome Isolation Kit, bacteria was used according to manufacturer's instructions. Briefly, 2 µg of total RNA samples was hybridized with prokaryotic rRNA-sequence-specific 5′-biotin-labelled oligonucleotide probes to selectively deplete large rRNA molecules from total RNA. Then, these rRNA-hybridized, biotinylated probes were removed from the sample with streptavidin-coated magnetic beads. The resulting RNA sample was concentrated using the RiboMinus concentrate module according to the manufacturer's protocol. The final RiboMinus RNA sample was subjected to thermal mRNA fragmentation using the Elute, Prime, Fragment Mix from the Illumina TruSeq RNA Sample Preparation Kit v2 (Low-Throughput protocol). The fragmented mRNA samples were subjected to cDNA synthesis using the Illumina TruSeq RNA Sample Preparation Kit (low-throughput protocol) according to the manufacturer's protocol. Briefly, cDNA was synthesized from enriched and fragmented RNA using SuperScript III reverse transcriptase (Invitrogen) and the SRA RT primer (Illumina). The cDNA was further converted into double-stranded DNA using the reagents supplied in the kit, and the resulting dsDNA was used for library preparation. To this end, cDNA fragments were end-repaired and phosphorylated, followed by adenylation of 3′ ends and adapter ligation. Twelve cycles of PCR amplification were then performed, and the library was finally purified with AMPure beads (Beckman Coulter) as per the manufacturer's instructions. A small aliquot (1 µl) was analysed on an Invitrogen Qubit and an Agilent Bioanalyzer. The bar-coded cDNA libraries were pooled at equal concentrations before sequencing on an Illumina HiSeq2000 using the TruSeq SR Cluster Generation Kit v3 and TruSeq SBS Kit v3. Data were processed with Illumina Pipeline software v1.82.
identifiers.namespace: GEO
identifiers.id: GSM3529945