|
| Status |
Public on Dec 01, 2017 |
| Title |
196062 |
| Sample type |
SRA |
| |
|
| Source name |
monoculture without mucin
|
| Organism |
Acinetobacter baumannii |
| Characteristics |
strain/background: ATCC 19606T
growth media: SB
|
| Growth protocol |
Twenty-five ml of SB or SB+M were inoculated with 0.25 ml of overnight SB cultures in 125-ml flasks, which were incubated at 37°C with shaking at 200 rpm for 4 h or until an OD600 of approximately 0.5 was reached. Triplicates of three different biological replicates of bacteria cultured in SB in the absence or presence of 0.5% mucin were used to isolate total RNA as described below.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Briefly, 10 ml of culture were directly added to an equal volume of acid phenol (pH 4.3 ± 0.2) and 0.1% SDS followed by immediate incubation at 90°C for 10 min. The aqueous fraction was then extracted with acid phenol:chloroform (5:1, pH 4.5 ± 0.2) followed by chloroform. Total RNA was precipitated overnight at -20°C with 2.5 volumes of absolute ethanol. RNeasy on-column DNase I treatment was performed as per the manufacturer’s instructions (Qiagen). DNA removal was confirmed using QuantiTect SYBER Green PCR kit (Qiagen) with primers 3968 and 3969 (Table S4) for the recA gene with amplification on a Bio-Rad CFX Connect real-time PCR detection system.
RNA quantity was routinely estimated using a NanoDrop ND-2000 spectrophotometer and quality was assessed with an Agilent Bioanalyzer 2100 using the RNA 6000 Pico LabChip Kit (Agilent). DNA-free total RNA was rRNA depleted by treatment with the Ribo-Zero Magnetic Kit (Epicentre). Depletion was assessed using the RNA 6000 Pico LabChip Kit and the Bioanalyzer 2100. Cognate rRNA-depleted technical triplicates isolated from each of the three independent biological samples cultured in SB or SB+M were combined to prepare six (three for each culture condition) Illumina-compatible libraries using the NEBNext Ultra Directional RNA Library preparation kit (New England Biolabs Inc.) according to the manufacturer’s instructions. All purifications were performed with Agencourt AMPure XP Beads (Beckman Coulter, Inc.) as described in the NEBNext protocol. After purification, the size of the DNA amplicons was assessed with the Agilent High Sensitivity DNA Kit on a Bioanalyzer 2100. The KAPA Library Quantification kit (Kapa Biosystems Inc.) was used to quantify libraries on a BioRad CFX Connect real-time PCR detection system.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
rRNA-depleted total RNA.
196062_S2_L001
Processed data file: 19606vs19606M1.txt
|
| Data processing |
Illumina MiSeq Generate FastQ 1.0.
edgeR values generated using CLC Genomics Workbench 7.0.4.
Genome_build: NC_009085.1, NC_009084.1, NC_009083.1
Supplementary_files_format_and_content: 19606vs19606M1.txt: Tab-delimited text file includes edgeR values for each Sample.
|
| |
|
| Submission date |
Jun 27, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Luis A Actis |
| E-mail(s) |
[email protected]
|
| Phone |
513-593-5422
|
| Organization name |
Miami University
|
| Department |
Microbiology
|
| Lab |
Actis
|
| Street address |
700 E. High St.
|
| City |
Oxford |
| State/province |
OH |
| ZIP/Postal code |
37129 |
| Country |
USA |
| |
|
| Platform ID |
GPL19442 |
| Series (1) |
| GSE100582 |
Mucin Acts as a Nutrient Source and a Signal for the Differential Expression of Genes Coding for Cellular Processes and Virulence Factors in Acinetobacter baumannii |
|
| Relations |
| BioSample |
SAMN07284856 |
| SRA |
SRX2962181 |
