|
| Status |
Public on Nov 28, 2020 |
| Title |
competition_amplicons_pilot_B |
| Sample type |
SRA |
| |
|
| Source name |
Bacteria
|
| Organism |
Bacillus subtilis |
| Characteristics |
strain: strains GLB115, GLB452, GLB434, GLB435, see Data S1 in paper for strain details
growth medium: Conditioned MCC medium with 1% glycerol (Parker et al, Cell Syt. 2020) with 10 uM IPTG.
|
| Growth protocol |
Overnight cultures started from fresh colonies were pooled and diluted to OD600 = 0.01. At OD600 =0.1, the pool 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 |
genomic DNA |
| Extraction protocol |
8 mL of culture (from the pool of barcoded strains in competition) was collected at OD600=0.1 and mixed with 0.8 mL of 10:1 ethanol/phenol stop solution by rapid inversion. Cells were pelleted at 3000 rcf at 4∘C for 10 min and the supernatant decanted. The cell pellets were stored at -80∘C until DNA extraction. To extract genomic DNA (gDNA), we used the Promega Wizard gDNA extraction kit, following the manufacturer’s protocol (scaling reagents volumes by 1/3×).
Amplicons were generated by two rounds of nested PCRs to append UMIs, multiplexing indices, and sequencing adapters.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
UMI collapsed barcode for each strain wer tallied and demultiplexed for PCR1 and PCR2 indices.
|
| Data processing |
Base calls performed using Casava version 1.7.
UMIs collapsed barcode counts for each strain in each condition (demultiplexed from PCR indices in the read) were counted using custom Matlab scripts. See Data S6 from paper for details on amplicon.
genome build: N/A
Library strategy: PCR amplicons with cell barcodes, UMIs, and multiplexing indices.
|
| |
|
| Submission date |
Nov 25, 2020 |
| Last update date |
Nov 28, 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 |
GPL21373 |
| Series (1) |
| GSE162169 |
Spurious regulatory connections dictate the expression-fitness landscape of translation factors |
|
| Relations |
| BioSample |
SAMN16913704 |
| SRA |
SRX9583195 |
