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| Status |
Public on Apr 11, 2018 |
| Title |
Mtb_EMB_4hrs_rep3 |
| Sample type |
SRA |
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| Source name |
M. tuberculosis H37Rv
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| Organism |
Mycobacterium tuberculosis H37Rv |
| Characteristics |
treatment: ethambutol
time point: 4hrs
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| Extracted molecule |
total RNA |
| Extraction 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Mtb.counts.txt
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| Data processing |
bcl2fastq2 Conversion Software
Trimming of Illumina adaptor sequences and low quality reads were done using Trimmomatic (v 0.33)
Quality trimmed read sequences were mapped to M. tuberculosis H37Rv Areference genomes using BWA (Cole et al. 1998; Stinear et al. 2008; Li et al. 2009). Gene expression estimates were made as raw read counts using the Pyhton script ‘HTSeq- count’ (model type – union). Counts data were converted to counts per million (cpm) and genes were filtered if they failed to achieve a cpm value of 1 in at least 30% of libraries per conditions.
Normalization of raw read counts and differential expression analysis was performed using DeSeq2
Genome_build: M. tuberculosis H37Rv
Supplementary_files_format_and_content: tab-delimited text files include read count values for each Sample
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| Submission date |
Dec 07, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
AMIT KUMAR SUBUDHI |
| E-mail(s) |
amit.subudhi@kaust.edu.sa
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| Phone |
+96540375986
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| Organization name |
King Abdullah University of Science and Technology
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| Department |
BESE
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| Lab |
Pathogen Genomics
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| Street address |
Level4, Builiding, 2, KAUST
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| City |
Thuwal |
| State/province |
Thuwal |
| ZIP/Postal code |
239556900 |
| Country |
Saudi Arabia |
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| Platform ID |
GPL17280 |
| Series (2) |
| GSE107831 |
Accelerating early anti-TB drug discovery by creating mycobacterial indicator strains that predict mode of action [Mycobacterium tuberculosis] |
| GSE107884 |
Accelerating early anti-TB drug discovery by creating mycobacterial indicator strains that predict mode of action |
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| Relations |
| BioSample |
SAMN08146669 |
| SRA |
SRX3456110 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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