|
| Status |
Public on Jan 16, 2020 |
| Title |
Whib6-3xfl (DespM)/MSP-espM Rep 1, part 2 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial Cell
|
| Organism |
Mycobacterium marinum |
| Characteristics |
strain: M strain (ATCC BAA-535)
genotype: espM deletion mutant, complemented
|
| Treatment protocol |
Cells were untreated
|
| Growth protocol |
M Marinum M based strains (Wild type, epsM mutant and complemented overexpressor strain) were cultured in 5mL 7H9 and 0.1% Tween-80 for two days, then moved to 25mL 7H9 and 0.1% Tween-80 for two days. All strains were then subcultured in Sauton’s media 0.01% Tween-80 at an OD of 0.8 for 48 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cultures were collected by centrifugation and stored at -80 for 24 hours. Cells were lysed using a bead beater. Total RNA was prepared from the pellets using the Qiagen RNeasy kit, as described by Bosserman et al., PNAS, 2017.
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 two lanes 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-multiplex and convert reads to FASTQ format
Reads from each lane were combined into individual files using standard UNIX commands
Trimmomatic (v0.33) 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 (v1.1.2) 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.
HTSeq software suite (v0.6.0) was used to analyze aligned and determine counts per gene feature in the M. tuberculosis CDC1551 genome
edgeR (v3.2) was used to normalize data by estimating effective library sizes using trimmed mean M-values
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 GLM functionality edgeR package
Genome_build: Mycobacterium marinum M reference genome (GCA_000018345.1)
Supplementary_files_format_and_content: Processed files are presented as .xlsx spreadsheets and represents the average of 2 biological replicates
|
| |
|
| Submission date |
Jul 29, 2019 |
| Last update date |
Jan 16, 2020 |
| 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 |
GPL21992 |
| Series (1) |
| GSE135072 |
EspM regulates gene expression in response to the ESX-1 system in Mycobacterium marinum |
|
| Relations |
| BioSample |
SAMN12392477 |
| SRA |
SRX6612110 |
