|
| Status |
Public on Apr 28, 2022 |
| Title |
Input_5 |
| Sample type |
SRA |
| |
|
| Source name |
Culture
|
| Organism |
Porphyromonas gingivalis |
| Characteristics |
treatment group: Input Pool
|
| Treatment protocol |
BALB/cJ mice were inoculated subcutaneously with either the library alone or in conjunction with either Streptococcus gordonii, or Fusobacterium necleatum. Bacteria were recovered and the transposon library was grown in vitro and passaged twice.
|
| Growth protocol |
A saturating transposon library using a Mariner-based transoposon was created by transposon mutagenesis in P. gingivalis.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was also isolated from the bacteria recovered from abscesses as well as from the input P. gingivalis library. DNA was shearded targeting 200-600 bp sing a Covaris sonicator. The sheared DNA was purified using a Qiagen PCR cleanup kit to remove te buffer. The DNA was used for C-tailing with Tdt and a mixture of dCTP and ddCTP optimized to give about 18 C residues. This reaction was cleaned up using a column and the product was used for PCR with a primer that binds inside the transposon near to the insertion site and a second poly G primer. The resulting PCR products (200-700 bp) contained the amplified DNA at the transposon insertion site and were then gel purified.
Library was constructed using a Nextera XT Kit (FC-121-1012).
|
| |
|
| Library strategy |
Tn-Seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
DM-05-Inp-5_S5
|
| Data processing |
Fastq files were downloaded from Illumina's BaseSpace for analysis.
Quality control (QC) of the raw sequence data was performed using FastQC (version 0.10.1) for each sequencing sample.
The majority of the trailing G's were removed at the ends of the sequences as well as the transposon sequence GAAGACCGGGGACTTATCATCCAACCTGTTA.
Trimmed reads were then aligned to the P. gingivalis genome ATCC 33277 (accession AP009380.1) using STAR aligner.
For the analysis using DESeq2, raw counts were obtained from the STAR aligned bam format files using HTSeq version 0.10.0
Genome_build: AP009380.1
Supplementary_files_format_and_content: tab-delimited text files with raw counts for each sample
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
|
| |
|
| Submission date |
Dec 11, 2021 |
| Last update date |
Apr 28, 2022 |
| Contact name |
Richard Lamont |
| E-mail(s) |
rich.lamont@louisville.edu
|
| Organization name |
University of Louisville
|
| Department |
Oral Immunology and Infectious Diseases
|
| Street address |
501 S. Preston St
|
| City |
Louisville |
| State/province |
Kentucky |
| ZIP/Postal code |
40292 |
| Country |
USA |
| |
|
| Platform ID |
GPL28580 |
| Series (1) |
| GSE190703 |
Porphyromonas gingivalis tyrosine kinase is a fitness determinant in polymicrobial infections |
|
| Relations |
| BioSample |
SAMN23899069 |
| SRA |
SRX13385101 |
