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| Status |
Public on Jun 13, 2020 |
| Title |
pUTE992 (atxA complementation) Rep1 |
| Sample type |
SRA |
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| Source name |
B. anthracis cell culture
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| Organism |
Bacillus anthracis |
| Characteristics |
strain: Ames ancestor
genotype: atxA-null, acpA-null, acpB-null, pUTE992 (atxA complementation)
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| Growth protocol |
Bacillus anthracis strains were cultivated at 37◦C in casamino acid medium containing 0.8% sodium bicarbonate (CA CO3). CA broth cultures were shaken at 200 r.p.m. in 5% atmospheric CO2. For strains harboring atxA, acpA, and acpB alleles under control of the hyperspank promoter (Phyperspank) (Britton 2002), expression was induced with isopropyl b-D-thiogalactoside (IPTG) during early exponential phase at 2 h and harvested at early stationary phase at 4 h. Optical densities for early exponential phase cultures ranged from OD600 0.25 to 0.35, and early stationary phase cultures ranged from 1.2 to 1.7.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Four ml samples were harvested at early stationary phase (OD600 5 1.2–1.7). Samples were centrifuged at 10,000 X g at 48◦C, and resulting pellets were resuspended in 500 μl of CACO3. An equivalent volume of saturated acid phenol (pH 4.3; Fisher Bioreagents, Fair Lawn, NJ) at 65◦C was added to each sample and transferred to screw top tubes containing 400 μl of 0.1 mm zirconia/silica beads (BioSpec Products, Bartlesville, OK). Samples were lysed mechanically for 1 min, incubated for 5 min at 65◦C and subsequently lysed for an additional 1 min followed by centrifugation at 3000 X g at 48◦C. Supernates were transferred to 500 μl of saturated acid phenol at 65◦C. Samples were vortexed, incubated at room temperature (RT) for 5 min and centrifuged at 16,000 X g for 3 min at 48◦C. One-third volume of chloroform was added to the aqueous phase. Following incubation for 10 min at RT, samples were centrifuged at 16,000 X g for 15 min at 48◦C. The aqueous phase was transferred to a new tube, and RNA was precipitated by the addition of one-half volume diethyl-pyrocarbonate (DEPC)-treated water and one total volume (aqueous phase + DEPC-treated water) of isopropanol followed by incubation at RT for 10 min. Samples were centrifuged at 16,000 X g for 15 min at 48◦C, and pellets containing precipitated RNA were washed with 75% ethanol. Following the removal of ethanol, pellets were air dried and finally resuspended in DEPC-treated water.
RNA libraries were prepared for sequencing using standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
pUTE992.vs.pUTE657.txt
pUTE992.vs.Ames.txt
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| Data processing |
Illumina Casava1.7 software used for basecalling.
The quality of the reads was assessed using FastQC (Andrews, 2010).
Reads were aligned to the B. anthracis strain ‘Ames Ancestor’, NCBI accession AE017334.2, using bowtie2 version 2.2.5 (Langmead and Salzberg, 2012) with default parameters.
The bedtools genomeCoverageBed command (Quinlan and Hall, 2010) was used to create bedgraph files from the aligned read files in BAM format. The IGVtools function of IGV (Thorvaldsdόttir et al., 2013) was used to convert the bedgraph files to tdf format files for use in IGV.
For differential gene expression analysis, sequencing files (BAM format) were imported into the Cufflinks pipeline. Triplicate sequencing results of each strain were compared to calculate Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) changes using the default setting. CuffDiff calculated log2(fold-change) in FPKM expression of genes between strains.
Genome_build: GCF_000008445.1_ASM844v1
Supplementary_files_format_and_content: Tab-delimited text files showing log2(fold-change) in expression between indicated strains for the complete Ames genome
Supplementary_files_format_and_content: tdf files showing genome coverage of reads along the genome per strain.
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| Submission date |
Jun 12, 2020 |
| Last update date |
Jun 13, 2020 |
| Contact name |
Theresa Marie Koehler |
| E-mail(s) |
Theresa.M.Koehler@uth.tmc.edu
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| Organization name |
McGovern Medical School
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| Department |
Microbiology and Molecular Genetics
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| Street address |
6431 Fannin Street, MSB 1.508
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77054 |
| Country |
USA |
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| Platform ID |
GPL28687 |
| Series (1) |
| GSE152357 |
Regulons and protein–protein interactions of PRD-containing Bacillus anthracis virulence regulators reveal overlapping but distinct functions |
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| Relations |
| BioSample |
SAMN15223291 |
| SRA |
SRX8537493 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4613424_pUTE992_1.bedgraph.tdf |
10.6 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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