|
| Status |
Public on Nov 24, 2021 |
| Title |
L_M9_inut_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
German Collection of Microorganisms and Cell Cultures GmbH
|
| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 |
| Characteristics |
sample type: input for mutational scanning - M9 media - replicate 2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Library preparation was carried out by using 50 ng of plasmid as template from the ‘input’ and ‘output’. For each library LIB1 (1-81 aa), LIB2 (73-155 aa) and LIB3 (155-227 aa), same region subjected to mutagenesis was amplified by PCR with specific primers that incorporated Nextera adapters: i) JVO18138/JVO18139 for LIB1, ii) JVO18140/JVO18141 for LIB2 and iii) JVO18142/JVO18143 for LIB3. The distribution of the library and concentration of library DNA was determined by DNA bioanalyzer and a DNA Qubit measurement respectively. Amplified DNAs from different libraries were pooled and sequenced on an Illumina MiSeq 2 x 300 bp at the next generation sequencing core unit of the Helmholtz Centre for Infection Biology (HZI).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
Around 30,000 transformants/library were obtained and pooled in 10 ml 1×PBS. Cells were washed twice on fresh 1×PBS prior to inoculation on the selective (M9+succinate) and non-selective (LB) media. The turbidity of the transformants suspension was measured and 4 ODs were stored as ‘input’. Next, 2 ODs were inoculated in 50 ml of either M9+succinate or LB in a 250 ml flask and incubated without aeration for 22h at 37°C. From the resulting cultures, 4 ODs were collected and considered ‘output’. Plasmid content from ‘input’ and ‘output’ was extracted by Nucleospin Mini plasmid kit (Macherey-Nagel) and used as template for library preparation for deep sequencing
Mutational Scanning - Results.xlsx
|
| Data processing |
The reads were used input for Enrich2 to determine enrichment of point mutations. For this, Enrich2 settings were set to include reads with an average read quality above 20 and up to 50 mismatches to the ProQ sequence. These results were then subsetted to
Genome_build: NC_016810.1
Supplementary_files_format_and_content: Mutational Scanning: counts of individual mutant (.xlsx); CoIP: coverage files (.wig)
|
| |
|
| Submission date |
May 16, 2021 |
| Last update date |
Nov 24, 2021 |
| Contact name |
Falk Ponath |
| E-mail(s) |
Falk.Ponath@helmholtz-hiri.de
|
| Organization name |
Helmholtz Institute for RNA-based Infection Research (HIRI)
|
| Street address |
Josef-Schneider-Straße 2
|
| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL22775 |
| Series (1) |
| GSE174509 |
Deep mutational scanning of RNA-binding protein ProQ suggests a mechanism of quality control by protease Lon |
|
| Relations |
| BioSample |
SAMN19228132 |
| SRA |
SRX10903038 |
