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Sample GSM1560345

Query DataSets for GSM1560345

Status

Public on May 20, 2015

Title

WT, Ethoxzolamide, pH 5.7, Rep A

Sample type

SRA

Source name

Bacterial cell_wildtype_Ethoxzolamide_pH5.7

Organism

Mycobacterium tuberculosis

Characteristics

strain: CDC1551
genotype: wildtype
ph: 5.7
treatment: Ethoxzolamide

Treatment protocol

Mtb cultures were resuspended in medium and examined under four conditions: 1) DMSO treated, pH 7.0, 2) DMSO treated, pH 5.7, 3) 40 µM ethoxzolamide treated, pH 7.0, and 4) 40 µM ethoxzolamide treated, pH 5.7. The CDC1551(phoP::Tn) mutant was grown in a similar manner and treated with DMSO at pH 7.0 and 5.7. Following six days incubation, total bacterial RNA was extracted. Two biological replicates were performed.

Growth protocol

Mtb strain CDC1551 was grown at 37C in T-25 vented, standing tissue culture flasks in 8 ml of buffered 7H9 medium seeded an initial OD of 0.2 Cultures were incubated at 37C for 6 days.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using the method Rohde, Abramovitch and Russell, Cell Host and Microbe, 2007. Briefly, RNA was stabilized and isolated using a guanidine thiocyanate-based lysis buffer as previously described. Pelleted mycobacteria were lysed using 5 μg/ml lysozyme, hot Trizol, and physical disruption with silica beads in a BeadBeater. Total RNA was isolated by chloroform extraction followed by direct application to QIAGEN RNeasy column purification. Residual DNA contamination was removed using Turbo DNAfree DNase (Ambion). RNA quality and integrity were examined using an Agilent Bioanalyzer prior to subjecting samples to rRNA depletion using the Epicentre Ribo-Zero depletion kit.
cDNA libraries were constructed using the Illumina TruSeq RNA library preparation kit (v2), omitting the polyA selection step. Multiplexing sequences are included in the title of the associated fastq.gz files.
After library quality control, sample libraries were pooled and sequenced in one lane of an Illumina HiSeq 2500 Rapid Run flow cell (v1) in 50bp, single-end read format (SE50). After the sequencing run, reads were de-multiplexed and converted to FASTQ format using the Illumina bcl2fastq (v1.8.4) script.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Illumina bcl2fastq (v1.8.4) was used to de-multiplexe and converte reads to FASTQ format
Trimmomatic (v0.30) was used to trim low quality bases and remove adapter sequences with a 4 bp sliding window, cutting when the read quality dropped below 15 or read length was less than 36 bp.
Bowtie was used to align trimmed reads to the M. tuberculosis CDC1551 genome (NCBI accession AE000516) with the -S option to produce SAM files as output.
SAMtools was used to calculate coverage depth (>98% coverage acrosss the genome for all samples).
HTSeq software suite was used to analyze aligned and determine counts per gene feature in the M. tuberculosis CDC1551 genome
DESeq was used to normalize data by estimating effective library sizes using robust regression
R was used for statistical analysis and differential gene expression by fitting a negative binomial model to each set of conditions and testing for differences utilizing the DESeq package
Qualimap was used to analyze coverage against the reference genome, sequence depth and pearson's correlation coefficient between replicates
Genome_build: M. tuberculosis CDC1551 genome (NCBI accession AE000516)
Supplementary_files_format_and_content: Processed files are presented as text files and represents genes that are induced or repressed >2 fold, with a p-value <0.05.

Submission date

Dec 05, 2014

Last update date

May 15, 2019

Contact name

Robert B. Abramovitch

Organization name

Michigan State University

Department

Microbiology and Molecular Genetics

Street address

567 Wilson Road