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| Status |
Public on Nov 28, 2020 |
| Title |
rend_CRISPRi_RF2 |
| Sample type |
SRA |
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| Source name |
Bacteria
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| Organism |
Bacillus subtilis |
| Characteristics |
strain: CRISPRi targeting RF2, CAG74815, lacA::Pxyl-dcas9(ErmR), amyE::Pveg-sgRNA_prfB(CmR)
growth medium: LB with 0.05% 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 |
5 mL of cell culture was added to 5 mL of cold (-30C) methanol, mixed by inversion and spun down at 3000 rcf for 10 min at 4C. The supernatant was decanted and the cell pellet frozen at -80C. RNA was extracted using the RNAeasy kit (QIAGEN).
Ribosomal RNA was depleted using the MICROBExpress kit (Thermo Fisher). The resulting RNA was fragmented for 25 s at 95C using RNA fragmentation reagents (Thermo Fisher, AM 8740). Fragments in the 15 to 45 nt range were selected, dephosphorylated at the 3’ end and ligated to a 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 5' and 3' ends of mapped reads between 15 and 45 nt in sizes were added separately at genomic positions to generate the wig file (thus leading to 4 wig files: 3’ forward, 3’ reverse, 5’ forward, 5’ reverse). Peak shadows were not removed.
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: Rend-Seq
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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 |
SAMN16913794 |
| SRA |
SRX9583136 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4944070_rend_CRISPRi_RF2_3_f.wig.gz |
2.4 Mb |
(ftp)(http) |
WIG |
| GSM4944070_rend_CRISPRi_RF2_3_r.wig.gz |
2.2 Mb |
(ftp)(http) |
WIG |
| GSM4944070_rend_CRISPRi_RF2_5_f.wig.gz |
2.4 Mb |
(ftp)(http) |
WIG |
| GSM4944070_rend_CRISPRi_RF2_5_r.wig.gz |
2.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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