title: GSM2882772: Mmar_CIP_4hrs_rep3; Mycobacterium marinum; RNA-Seq
library_construction_protocol: RNA isolation was performed with a NucleoSpin® RNA kit (Machery Nagel). In total 25 OD units were spun down and used for isolation per sample. Isolation was performed with a biological triplicate. There was one deviation to the first step of the protocol to lyse the mycobacterial cells. Cells were disrupted by 0.1 mm zirconium beads in 500 μL Buffer RA1 and 5 μL ß-mercaptoethanol for 1 minute. Subsequent steps were according to protocol provided by the manufacturer. After RNA isolation, RNA concentrations were measured. RNA integrity numbers (RIN) were determined with a bioanalyzer 2100 expert (Agilent). The RIN value was measured for one sample per triplicate. All RIN values were >9. As ribosomal RNA comprises the vast majority of the extracted RNA population, depletion of these molecules through RiboMinus-based rRNA depletion was used in efforts to increase the coverage of mRNA and to reduce rRNA reads. For this mRNA enrichment, the Invitrogen's RiboMinusTM Prokaryotic kit was used according to manufacturer's instructions. Briefly, 2 µg of total RNA samples was hybridized with prokaryotic rRNA sequence-specific 5′-biotin labeled 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 RiboMinusTM concentrate module according to the manufacturer's protocol. The final RiboMinusTM RNA sample was subjected to thermal mRNA fragmentation using Elute, Prime, and fragment Mix from the Illumina TruSeqTM RNA sample preparation kit v2 (Low-Throughput protocol). The fragmented mRNA samples were subjected to cDNA synthesis using the Illumina TruSeqTM RNA sample preparation kit (Low-Throughput protocol) according to manufacturer's protocol. Briefly, cDNA was synthesized from enriched and fragmented RNA using SuperScript III Reverse Transcriptase (Invitrogen) and 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 analyzed on Invitrogen Qubit and Agilent Bioanalyzer. The bar-coded cDNA libraries were pooled together in equal concentrations in one pool before sequencing on Illumina HiSeq2000 using the TruSeq SR Cluster Generation Kit v3 and TruSeq SBS Kit v3. Data were processed with the Illumina Pipeline Software v1.82.
attributes.tag: GEO Accession
attributes.value: GSM2882772