|
| Status |
Public on Dec 05, 2017 |
| Title |
ribo_footprints_mazF_5m |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial liquid culture
|
| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655 delta_mazF
plasmid: pBAD30-mazF
time induction: 5 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%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by filtration and were scraped into liquid nitrogen. Lysis was conducted on a mixer mill (Qiagen) and lysate was treated with MNase and separated on a sucrose gradient to yield ribosome protected mRNA fragments. RNA was harvested by phenol chloroform extraction.
See publication for details. RNA fragments were size-selected, dephosphorylated, and ligated to a linker RNA. RNA was then reverse transcribed using an oligo recognizing the ligated linker. cDNA was circularized and cDNA arising from rRNA was subtracted using custom oligos. Finally, libraries were PCR amplified and sequenced.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Ribosome protected mRNA
ribosome_footprint_ends.csv.gz
|
| Data processing |
Library strategy: Ribo-Seq
Single-end reads were mapped using bowtie2 default settings after trimming off adapter sequences.
A single count was added at the 5' end or 3' end of reads.
Samples were sequencing depth normalized using by calculating reads per million mapped.
Genome_build: NCBI reference sequence: NC_000913.2
Supplementary_files_format_and_content: ribosome_footprint_ends.csv.gz uniquely mapping 5' and 3' end footprints are recorded for each sample at all positions in the genome. Counts are sequencing-depth normalized using reads per million mapped.
|
| |
|
| 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 |
GPL21222 |
| Series (2) |
| GSE107328 |
E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis [Ribo-Seq] |
| GSE107330 |
E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis |
|
| Relations |
| BioSample |
SAMN08097777 |
| SRA |
SRX3421854 |
