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| Status |
Public on Dec 05, 2017 |
| Title |
MazF_totalRNA_5m_rep1 |
| Sample type |
SRA |
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| Source name |
Bacterial liquid culture
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| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655 delta_mazF
plasmid: pBAD30-MazF
ribozero: No
ercc spike-in: Prior to extraction
time induction: 5 minutes
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| 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.
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| 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, samples with rRNA subtraction with treated with bacterial Ribo-Zero kit (Illumina). Next, RNA was fragmented with RNA fragmentation reagents (Ambion). First strand cDNA synthesis was conducted using random primers and Superscript III (Invitrogen). To enable strand specificity, second strand synthesis was conducted using dUTP instead of dTTP with RNase H, E. coli DNA ligase, and E. coli DNA polymerase. Ends were repaired with T4 DNA polymerase, Klenow DNA polymerase, and T4 PNK. 3’ ends were adenylated with the Klenow fragment (3’>5’ exo-). Y-shaped adapters were ligated and 9-12 cycles of PCR were conducted with standard Illumina primers with multiplexing indexes. Libraries were extracted from an acrylamide gel and submitted for sequencing at the MIT BioMicro Center using standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
[processed data file: ]
total_RNA_rpm.csv.gz
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| Data processing |
Paired-end reads mapped to MG1655 genome using bowtie2 with default settings. Any adapter sequences were removed prior to mapping.
For each uniquely mapped fragment (ribozero treated samples) or all mapped fragments (total RNA samples), at all aligned positions a count was added.
Counts were depth normalized to a pseudo-reference sample calculated from all samples (ribozero treated samples) or using counts per million counts (total RNA samples).
For ribozero treated samples, counts at all positions were log2 transformed and a cleavage ratio was calculated (+ MazF - empty vector) for assessing MazF cleavage in transcripts.
Genome_build: NCBI reference sequence: NC_000913.2
Supplementary_files_format_and_content: 5m_ribozero_counts_cleavage_ratio.csv.gz is a log-transformed list of sequencing depth-normalized counts for every genomic position at both strands for each ribozero treated sample as well as the average cleavage ratio calculated from all samples. total_RNA_rpm.csv.gz is a list of sequencing depth-normalized counts for every genomic position at both strans for all total RNA samples.
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| Submission date |
Nov 24, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Culviner |
| Organization name |
MIT
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| Department |
Biology
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| Lab |
Laub
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| Street address |
31 Ames St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL21222 |
| Series (2) |
| GSE107327 |
E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis [RNA-seq] |
| GSE107330 |
E. coli MazF does not create specialized ribosomes that translate leaderless mRNAs, but instead blocks rRNA maturation and ribosome biogenesis |
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| Relations |
| BioSample |
SAMN08097784 |
| SRA |
SRX3421864 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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