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Sample GSM3529944

Query DataSets for GSM3529944

Status

Public on Dec 25, 2018

Title

MVU_KOWhiB6_1

Sample type

SRA

Source name

M. marinum MVU WhiB6 mutant

Organism

Mycobacterium marinum M

Characteristics

tissue: M. marinum MVU WhiB6 mutant
genotype/variation: WhiB6 mutant

Extracted molecule

total RNA

Extraction 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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

processed data file: VU_EmptyVector_KnockOut.txt

Data processing

bcl2fastq2 Conversion Software
Trimming of Illumina adaptor sequences and low quality reads were done using Trimmomatic (v 0.33)
For the wild type and ESX1 mutants comparision, the Illumina reads were mapped with SMALT (default parameters) against the M. tuberculosis H37Rv or M. marinum M reference. From the read count, which was obtained with bedtools, parameter multicov, with -D to include duplicates and -q 5 to exclude repetitive mapping reads, we performed a differential expression analysis with DESeq using default parameters. For the whib6 knockout and complementary comparision, we mapped the trimmed reads back to the same reference genome with bowtie2 and the differently expressed genes were called by cuffdiff2.
Normalization of raw read counts and differential expression analysis was performed using DeSeq or CuffDiff2
Supplementary_files_format_and_content: tab-delimited text files include read count values for each Sample

Submission date

Dec 24, 2018

Last update date

Dec 27, 2018

Contact name

Qingtian Guan

E-mail(s)

[email protected]

Organization name

King Abdallah University of Science and Technology

Street address

Thuwal