|
| Status |
Public on Jul 02, 2021 |
| Title |
DMSO_1: WT treated with DMSO, pH 5.7 rep 1 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cell
|
| Organism |
Mycobacterium tuberculosis CDC1551 |
| Characteristics |
strain: CDC1551
treatment: DMSO
acidity: pH 5.7
|
| Treatment protocol |
Mtb cultures were subsequently treated with 20 µM AC2P20. For the control, cultures treated with an equal amount of DMSO. Following a 24 h incubation period, RNA was isolated from the bacterial cultures and used for RNA-seq. For each condition, 2 biological replicates were performed.
|
| Growth protocol |
Mtb cultures were grown to an OD600 of 0.5 in 8 mL of 7H9 OADC buffered media (pH 5.7) at 37°C and 5% CO2 in standing T-25 culture flasks.
|
| 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 determin counts per gene feature in the M. tuberculosis CDC1551 genome
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 edgeR 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 .csv spreadsheets and represents the average of 2 biological replicates
|
| |
|
| Submission date |
Jun 05, 2020 |
| Last update date |
Jul 02, 2021 |
| Contact name |
Robert B. Abramovitch |
| Organization name |
Michigan State University
|
| Department |
Microbiology and Molecular Genetics
|
| Street address |
567 Wilson Road
|
| City |
East Lansing |
| State/province |
MI |
| ZIP/Postal code |
48824 |
| Country |
USA |
| |
|
| Platform ID |
GPL20114 |
| Series (1) |
| GSE151884 |
AC2P20 selectively kills M. tuberculosis at acidic pH by depleting free thiols |
|
| Relations |
| BioSample |
SAMN15146099 |
| SRA |
SRX8479814 |
