|
| Status |
Public on Dec 05, 2017 |
| Title |
5'OH_mazF_20m |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial liquid culture
|
| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655
plasmid: pBAD33-mazF
time induction: 20 minutes
|
| Growth protocol |
Separate E. coli colonies were picked from LB agar plates for each replicate. Overnight cultures (12-16 hrs) were grown at 37°C in M9 media supplemented with 0.1% casamino acids, 0.4% glycerol, 0.4% glucose, 2 mM MgSO4, and 0.1 mM CaCl2. Overnight cultures were back diluted into fresh media and grown 3-4 hours to an OD600 of ~0.35 at 37°C in an orbital shaker at 200 RPM. Cultures were centrifuged, washed, and back diluted into media lacking glucose. After at least 30 minutes of recovery Para was induced by adding arabinose to 0.2%. Cells were harvested by centrifuging 1 mL of culture for 1 minute at maximum speed on a benchtop centrifuge. Pellets were immediately flash frozen in lN2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol (Invitrogen) and Direct-zol RNA MiniPrep kit (Zymo).
See publication for complete protocol. Briefly, 5’-P terminated RNA was degraded with terminator exonuclease (Epicentre) and then 5’-OH terminated RNA was phosphorylated with optikinase (USB). An RNA linker containing an amplification region was then ligated onto phosphorylated RNA. Reverse transcription was conducted with a primer with a randomized region as well as an amplification region. Finally, libraries were amplified and sequenced.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
5OH_counts_and_ratio.csv.gz
|
| Data processing |
Library strategy: 5'OH-seq
Single-end reads were mapped using bowtie2 default settings.
A single count was added at the 5' end of all reads.
Samples were sequencing depth normalized using by calculating reads per million mapped.
A 5'-OH ratio was determined by log transforming the + MazF to empty vector ratio.
Genome_build: NCBI reference sequence: NC_000913.2
Supplementary_files_format_and_content: 5OH_counts_and_ratio.csv.gz contains single-nucleotide mapping of read 5' ends after reads per million correction for both empty vector and MazF as well as the log2 5'OH ratio (+ mazF / empty vector) calculated at each position
|
| |
|
| Submission date |
Nov 24, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Culviner |
| Organization name |
MIT
|
| Department |
Biology
|
| Lab |
Laub
|
| Street address |
31 Ames St
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL14548 |
| Series (2) |
| GSE107329 |
E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis [5'OH-seq] |
| GSE107330 |
E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis |
|
| Relations |
| BioSample |
SAMN08097734 |
| SRA |
SRX3421852 |
