|
| Status |
Public on May 06, 2021 |
| Title |
PBP1a 16-Hour Depletion rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cells
|
| Organism |
Agrobacterium fabrum str. C58 |
| Characteristics |
genotype: {delta}tetRA::mini-Tn7-GM-Plac-pbp1a
treatment: Washed of IPTG (3x)
depletion time: 16 hours
|
| Treatment protocol |
Cells were pelleted by centrifugation at 7000 x g for 5 minutes. Cell pellets were washed three times with ATGN by centrifugation and resuspension to remove IPTG. PBP1a depletions were grown in the absence of IPTG for 6 hours and 16 hours. WT + IPTG and PBP1a + IPTG were grown with 1mM IPTG added back.
|
| Growth protocol |
Cells were grown overnight in 2 ml of ATGN minimal media at 28°C with shaking; the + PBP1a strains and WT + IPTG strains were supplemented with 1mM IPTG.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated from the -PBP1a_6hr strains after 6 hours of growth, and RNA was isolated from all other strains after 16 hours of growth. To prepare samples, a culture volume equivalent to 6 ml at an optical density at 600 nm (OD600) of 0.2-0.3 was pelleted by centrifugation at 7000 x g for 5 minutes and pellets were resuspended in 1mL of ATGN media and incubated with 2 mL of RNAProtect reagent (QIAgen) for 15 min at room temperature. Cells were lysed with 10 mg lysozyme, and RNA was extracted using the QIAgen RNEasy kit.
DNA libraries for sequencing were constructed following the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq mRNA stranded sample preparation kit. The sample concentration was determined by Qubit flourometer (Invitrogen) using the Qubit HS RNA assay kit, and the RNAintegrity was checked using the Fragment Analyzer automated electrophoresis system. Briefly, the poly-A containing mRNA is purified from total RNA, RNA is fragmented, double-stranded cDNA is generated from fragmented RNA, and the index containing adapters are ligated to the ends. The amplified cDNA constructs were purified by addition of Axyprep Mag PCR Clean-up beads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Sample 18
|
| Data processing |
cutadapt version 0.16 was used to remove detected adapter sequences.
bowtie2 (2.3.4.3) was used to purge all samples of reads that mapped to transcripts are rRNA genes.
The remaining reads were mapped to the Agrobacterium fabrum str. C58 genome using STAR (version 2.5.4b)
Bioconductor package DESeq2 was used for pairwise comparisons showing differential expression of genes
Genome_build: Agrobacterium fabrum str. C58 v1
Supplementary_files_format_and_content: Excel Workbook file of pairwise comparisons showing differential expression in each condition
|
| |
|
| Submission date |
May 05, 2021 |
| Last update date |
May 06, 2021 |
| Contact name |
Jacob Matthew Bouchier |
| Organization name |
University of Missouri
|
| Department |
Division of Biological Sciences
|
| Lab |
Pam Brown Lab
|
| Street address |
105 Tucker Hall
|
| City |
Columbia |
| State/province |
Missouri |
| ZIP/Postal code |
65201 |
| Country |
USA |
| |
|
| Platform ID |
GPL30075 |
| Series (1) |
| GSE173921 |
Decreased Peptidoglycan Synthesis Induces the RSI Invasion Switch in Agrobacterium tumefaciens |
|
| Relations |
| BioSample |
SAMN19023197 |
| SRA |
SRX10783486 |
