|
| Status |
Public on Nov 27, 2017 |
| Title |
Wild Type, Rep B |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial Cell
|
| Organism |
Mycobacterium marinum |
| Characteristics |
strain: M strain (ATCC BAA-535)
genotype: Wild Type
|
| Treatment protocol |
n/a
|
| Growth protocol |
M. marinum cultures were grown to saturation in 7H9 defined broth and diluted to an OD600 of 0.8 in 50 ml of Sauton's defined broth with 0.05% Tween 80. After 48 h of growth at 30°C, cells were harvested by centrifugation. Whole-cell lysates were generated by lysing the mycobacterial cells with a Mini-Bead Beater-24 (BioSpec).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using an RNeasy minikit (Qiagen) according to the manufacturer's recommendation with the following modifications. After bacterial cells were harvested, they were resuspended in 1 ml of RLT buffer containing 0.1% 2-mercaptoethanol and transferred to a 2-ml screw-cap tube containing glass beads. The mixture was homogenized in a mini-bead beater (BioSpec Products) with three 30-s pulses. RNA samples were treated with DNase I. Total RNA quality was assessed using an Agilent Bioanalyzer 2100 and the Agilent RNA 6000 Nano Reagent kit (16S rRNA chip) in the University of Notre Dame Genomics Core Facility.
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 |
Jun 02, 2017 |
| 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
|
| City |
East Lansing |
| State/province |
MI |
| ZIP/Postal code |
48824 |
| Country |
USA |
| |
|
| Platform ID |
GPL21992 |
| Series (1) |
| GSE99632 |
WhiB6 regulation of ESX-1 gene expression is controlled by a negative feedback loop in Mycobacterium marinum |
|
| Relations |
| BioSample |
SAMN07190155 |
| SRA |
SRX2881195 |
