|
| Status |
Public on Aug 05, 2021 |
| Title |
CAMHB MEM 2 |
| Sample type |
SRA |
| |
|
| Source name |
E. coli K12 MG1655
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
media: CAMHB
antibiotic: meropenem (256ug/mL)
|
| Treatment protocol |
Total RNA was sampled from duplicate cultures. Strains were cultured overnight in the respective media as described above. Mid-log phase cultures were diluted (starting OD600~0.05) into 30mL media for untreated conditions, or 30 mL media containing the MIC90 of the respective antibiotic for the respective media. Flasks were incubated for 30 min at 37C with shaking. Cell broth was centrifuged and supernatant was removed.
|
| Growth protocol |
Escherichia coli strain MG1655 was grown in three media: (1) M9 minimal medium (47.8 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 18.7 mM NH4Cl, 2 mM MgSO4 and 0.1 mM CaCl2) supplemented with 0.2% (w/v) glucose (M9); (2) Roswell Park Memorial Institute 1640 (RPMI) (Thermo Fisher Scientific) supplemented with 10% LB (R10LB); (3) Mueller Hinton Broth (Sigma-Aldrich) supplemented with 25 mg/L Ca2+ and 12.5 mg/L Mg2+ (CA-MHB). Glycerol stocks of E. coli were streaked onto Luria Broth agar (LB Agar) and grown at 37°C overnight.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After inoculation and growth, 3 mL of cell broth (OD600 ~ 0.5) was immediately added to 2 volumes Qiagen RNA-protect Bacteria Reagent (6 mL), vortexed for 5 seconds, incubated at room temperature for 5 min, and immediately centrifuged for 10 min at 17,500 RPMs. The supernatant was decanted, and the cell pellet was stored in the -80°C. Cell pellets were thawed and incubated with Readylyse Lysozyme, SuperaseIn, Protease K, and 20% SDS for 20 minutes at 37°C. Total RNA was isolated and purified using the Qiagen RNeasy Mini Kit columns and following vendor procedures. An on-column DNase-treatment was performed for 30 minutes at room temperature. RNA was quantified using a Nano drop and quality assessed by running an RNA-nano chip on a bioanalyzer. The rRNA was removed using Illumina Ribo-Zero rRNA removal kit for Gram Negative Bacteria. A KAPA Stranded RNA-Seq Kit (Kapa Biosystems KK8401) was used following the manufacturer’s protocol to create sequencing libraries with an average insert length of around ~300 bp.
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Raw sequencing reads were mapped to the reference genome (NC_000913.3) using bowtie v1.1.2 with parameters -X 1000 -n 2
Transcript abundance was quantified using summarizeOverlaps from the R GenomicAlignments package, with strand inversion for the dUTP protocol and “IntersectionStrict” mode
Transcripts per Million (TPM) were calculated using DESeq2’s fpkm function, then normalizing for library size.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: Comma-separated text files include log-TPM (log2-transformed Transcripts per million) for each sample
|
| |
|
| Submission date |
Oct 13, 2020 |
| Last update date |
Aug 05, 2021 |
| Contact name |
Anand Sastry |
| E-mail(s) |
avsastry@eng.ucsd.edu
|
| Organization name |
University of California, San Diego
|
| Department |
Bioengineering
|
| Lab |
Systems Biology Research Group
|
| Street address |
9500 Gilman Dr
|
| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL21117 |
| Series (1) |
| GSE159494 |
Expression profiling of E. coli in three media treated with antibiotics |
|
| Relations |
| BioSample |
SAMN16432197 |
| SRA |
SRX9288053 |
