|
| Status |
Public on Jan 17, 2020 |
| Title |
Constutive clbQ expressing variant |
| Sample type |
SRA |
| |
|
| Source name |
RNA-seq
|
| Organism |
Escherichia coli |
| Characteristics |
strain: M1/5
genotype: +pBAD24-tetAP-clbQ-rrnB
|
| Treatment protocol |
3.36 mL of pooled bacterial culture was mixed with 0.42 mL stop solution (95 % ethanol, 5 % phenol).
|
| Growth protocol |
Bacteria where grown in Greiner 96-well Flat Bottom white polystyrol plates in 150µl M9-medium with 1g L‑1 casein hydrolysate, using a Tecan Infinite 200 Reader (Tecan Group), 37°C, shaking for 10min with amplitude 2 in 15min intervals. Bacterial cultures with OD600nm of 0.4 (three biological triplicates with eight technical replicates, 140µl each), were pooled and harvested in late exponential growth phase by centrifugation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The suspension was centrifuged (5 min, 4 °C) and the bacterial pellet was resuspended in 35 µL lysis buffer (lysozyme in TE buffer, 85 mg/mL). After 10 min incubation at RT and gentle vortexing every 10 s, 1 mL TRizol ® (Thermo Fisher Scientific) was added and the isolation process was continued according to the manufacturer's protocol. 10 µg of isolated total RNA were used for DNA digestion, using the Turbo DNase (Turbo-DNA-free™ Kit, Thermo Fisher Scientific) according to the manufacturer's instructions.
The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified (11 PCR cycles) to about 10-20 ng/µl using a high fidelity DNA polymerase. The TruSeq barcode sequences AGGATAGG or TCAGAGCC, which are part of the 5' TruSeq sequencing adapter were used. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
total RNA
|
| Data processing |
Raw sequence reads were quality checked using quality control tool FastQC.
Reads were mapped using bwa with standard parameters (doi: 10.1093/bioinformatics/btp324)
Further analysis were carried out using ReadXplorer 2.2.3 (doi: 10.1093/bioinformatics/btw541)
Genome_build: The reads were mapped to the genome sequence of E. coli M1/5. The genome sequence is currently been submitted (accession number is pending).
Supplementary_files_format_and_content: Excel files including TPM values for each sample
|
| |
|
| Submission date |
Jan 16, 2020 |
| Last update date |
Jan 17, 2020 |
| Contact name |
Ulrich Dobrindt |
| E-mail(s) |
ulrich.dobrindt@ukmuenster.de
|
| Phone |
+492519802875
|
| Organization name |
University of Münster
|
| Department |
Institute of Hygiene
|
| Street address |
Mendelstrasse 7
|
| City |
Muenster |
| State/province |
NRW |
| ZIP/Postal code |
48149 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21222 |
| Series (1) |
| GSE143807 |
Influence of the putative transcription factor ClbR and the thioesterase ClbQ on colibactin synthesis regulation and characterization of critical transcription start sites in Escherichia coli |
|
| Relations |
| BioSample |
SAMN13873370 |
| SRA |
SRX7572193 |
