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| Status |
Public on Nov 28, 2020 |
| Title |
ribo3_GLB452 |
| Sample type |
SRA |
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| Source name |
Bacteria
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| Organism |
Bacillus subtilis |
| Characteristics |
strain: GLB452, see Data S1 in paper for strain details
growth medium: Conditioned MCC medium (Parker et al, Cell Syst. 2020) with 1% glycerol, 10 uM IPTG, 0.5% w/v xylose
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| Growth protocol |
Overnight cultures were diluted to OD600 0.0003 in 250 mL fresh media. The culture was kept in a 2.8 L flask at 37C with aeration (200 rpm) until OD600 reached between 0.15 to 0.3.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction was performed as described in detail previously (Li et al., Cell 2014 and Lalanne et al, 2018). 250 mL of cell culture was rapidly filtered at 37C by passing through a nitrocellulose filter. Cell pellets was were rapidly collected using a pre-warmed metal table crumber, flash frozen in liquid nitrogen, and combined with frozen droplets of lysis buffer. Cells and lysis buffer were pulverized in 10 mL canisters (Retsch) pre-chilled in liquid nitrogen using Qiagen TissueLyser II. Pulverized lysate was thawed on ice and clarified by centrifugation at 4C.
Ribosome protected mRNA fragments were size selected via gel purification, dephosphorylated at the 3’ end and ligated to 5' adenylated DNA oligo. After reverse transcription, the single stranded DNA was circularized, and PCR amplified.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Sequence reads were trimmed for adaptor sequences.
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| Data processing |
Base calls performed using Casava version 1.7.
Trimmed reads were aligned to NC_000964.3 using Bowtie v1.0.1 with options -v 1 -k 1. To deal with non-template addition during reverse transcription, reads with a mismatch at their 5' end had their 5' end re-assigned to the immediate next downstream position. The footprint reads with size between 15 to 44 nucleotides in length were mapped to the genome using the center-weighted approach: for each footprint read, the center residues that are at least 10 nucleotides away from either ends were given a weight of one over the length of the fragment (minus the 10 nucleotides on both ends) to generate the wig file, for footprint <21 nt long, the read count of 1 was added at the center of the footprint.
genome build: NC_000964.3
processed data files format and content: wiggle file with two columns: first column containing chromosome positions and second column containing the number of reads mapped to the position.
Library strategy: Ribo-Seq (ribosome protected mRNA)
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| Submission date |
Nov 25, 2020 |
| Last update date |
Nov 29, 2020 |
| Contact name |
Jean-Benoit Lalanne |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Department |
Genome Sciences
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| Lab |
Jay Shendure
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| Street address |
3720 15th Ave NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL24109 |
| Series (1) |
| GSE162169 |
Spurious regulatory connections dictate the expression-fitness landscape of translation factors |
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| Relations |
| BioSample |
SAMN16913785 |
| SRA |
SRX9583145 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4944079_ribo3_GLB452_f.wig.gz |
1.1 Mb |
(ftp)(http) |
WIG |
| GSM4944079_ribo3_GLB452_r.wig.gz |
1.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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