|
| Status |
Public on Nov 28, 2020 |
| Title |
RNAseq_E3_D4_GLB426 |
| Sample type |
SRA |
| |
|
| Source name |
Bacteria
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: GLB426, see Data S1 in paper for strain details
growth medium: Conditioned MCC medium with 1% glycerol (Parker et al, Cell Syt. 2020) with inducers. See Data S3 from paper for details.
|
| Growth protocol |
Overnight cultures started from fresh colonies were diluted to OD600 = 0.01. At OD600 =0.1, cultures were diluted to OD600 = 0.0001 in 25 mL pre-warmed medium. The culture was kept in a 125 mL flask at 37C with aeration (200 rpm) until OD600 reached 0.3.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 mL of culture were collected and mixed with 1 mL of 10:1 ethanol/phenol stop solution by rapid inversion. Cells were pelleted at 3000 rcf for 10 min at 4C. The supernatant was decanted and cell pellets stored at -80C until RNA extraction. RNA was extracted using the RNeasy plus kit with gDNA eliminator column (Qiagen) following manufacturer’s instructions.
Ribosomal RNA was depleted using the MICROBExpress kit (Thermo Fisher). The resulting RNA was fragmented for 1 min 45 s at 95C using RNA fragmentation reagents (Thermo Fisher, AM 8740), dephosphorylated at the 3’ end and poly-A tailed using T4 PNK (NEB). Revese transcription was performed using indexed poly-dT primer using SSIII (Thermo Fisher). After reverse transcription, cDNA in the range 100 to 120 nt were size selected, and ligated to an adapter with T4 DNA ligase (NEB). The ligated cDNA was size selected (135 to 155 nt) and PCR amplified.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Sequence reads were trimmed for poly A stretches at the 3’ ends.
|
| Data processing |
Base calls performed using Casava version 1.7.
Poly A tails were stripped, and the resulting reads aligned to the NC_000964.3 using Bowtie v1.0.1 with options -v 1 -k 1, and the 3’ ends of mapped reads were summed at each genomic position.
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.
|
| |
|
| Submission date |
Nov 25, 2020 |
| Last update date |
Nov 29, 2020 |
| Contact name |
Jean-Benoit Lalanne |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Washington
|
| Department |
Genome Sciences
|
| Lab |
Jay Shendure
|
| Street address |
3720 15th Ave NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
| |
|
| Platform ID |
GPL24109 |
| Series (1) |
| GSE162169 |
Spurious regulatory connections dictate the expression-fitness landscape of translation factors |
|
| Relations |
| BioSample |
SAMN16913747 |
| SRA |
SRX9583155 |
