|
| Status |
Public on Dec 19, 2023 |
| Title |
Input_2 |
| Sample type |
SRA |
| |
|
| Source name |
Escherichia coli MG1655
|
| Organism |
Escherichia coli |
| Characteristics |
strain: MG1655 transformed with pCB453
treatment: untreated
|
| Treatment protocol |
The culture grown in LB was sampled and washed twice with M9 minimal medium to remove traces of the LB medium, to reach OD600 0.01 in 50 ml M9 minimal medium (1x M9 salts, 1 mM thiamine hydrochloride, 0.4% glucose, 0.2% casamino acids, 2 mM MgSO4, 0.1 mM CaCl2). The culture was incubated at 37°C with shaking until it reached OD600 1, allowing ~6 replications.
|
| Growth protocol |
E. coli strain MG1655 was initially transformed with a dCas9 encoding plasmid (2.0 kV, 200 Omega, and 25 μF). The resulting strain SG332 was transformed with the sgRNA library by electroporation and recovered in 900 µl SOC for 1.5h at 37 °C with shaking at 250 rpm. Different dilutions of the recovered cells were plated on Luria Bertani (LB) agar containing the 34 µg/mL chloramphenicol and incubated for 16 h to check the number of the resulting colonies (~56^5 colonies). The recovered culture was back-diluted to OD600 0.01 in LB medium (10 g/L NaCl, 5 g/L yeast extract, 10 g/L tryptone) with 34 µg/mL chloramphenicol and incubated at 37 °C with shaking for 13h.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5 mL of the culture was sampled before (as input) and after being incubated in M9 minimal medium (at OD600 0.2, 0.6 and 1). The plasmids were extracted by miniprep (Nucleospin Plasmid, Macherey-Nagel).
The sequencing library was generated using the KAPA HiFi HotStart Library Amplification Kit for Illumina® platforms (Roche)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
purine_screen_logFC.csv
|
| Data processing |
Library strategy: DNA-seq
Paired-end reads were merged using BBMerge (version 38.69) with parameters “qtrim2=t, ecco, trimq=20, -Xmx1g”
Sequence reads with perfect matches were assigned to the gRNA library using a Python script
After filtering by 1 count per million in minimal 4 samples, read counts of each gRNA were normalized by scale factors determined from non-targeting guides using the trimmed mean of M-values method in edgeR (version 3.28.0). An extra column was added to the design matrix to account for batch effects between the two cultures. Differential abundance (logFC) of gRNAs between time points and that of the input library were calculated using a quasi-likelihood F test after fitting a generalized linear model.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: CSV, normalized differential abundance of each gRNA in different time points
|
| |
|
| Submission date |
Feb 16, 2022 |
| Last update date |
Dec 19, 2023 |
| Contact name |
Yanying Yu |
| E-mail(s) |
[email protected]
|
| Organization name |
Helmholtz Institute for RNA-based Infection Research
|
| Lab |
INTEGRATIVE INFORMATICS FOR INFECTION BIOLOGY
|
| Street address |
Josef-Schneider-Str. 2 / D15
|
| City |
Würzburg |
| State/province |
DE Deutschland |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL25368 |
| Series (1) |
| GSE196911 |
Improved prediction of bacterial CRISPRi guide efficiency from depletion screens through mixed-effect machine learning and data integration |
|
| Relations |
| BioSample |
SAMN25999566 |
| SRA |
SRX14206101 |
