|
| Status |
Public on Jan 10, 2023 |
| Title |
RpoD mutatnt Culture 23oC [rpoD_3_C_23_S20] |
| Sample type |
SRA |
| |
|
| Source name |
Bacteria
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype: RpoD D445V mutant
treatment: Grown at 23oC
|
| Treatment protocol |
E. coli was grown in LB broth at 37C or 23C
|
| Growth protocol |
MG1655 WT or RpoD D445V mutant E. coli were grown at 37C or 23C to mid log phase (OD600 ~ 0.5-0.6)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using method II of (Hinton 1989)
rRNA subtraction was performed with the bacterial RiboMinus Kit (Ambion) according to manufacturer instructions. cDNA was prepared using the NEBNext strand specific kit (New England BioLabs) according to manufacturer instruction for libraries with 300-450 bp insert size with the following modifications. Illumina adaptors sequences based on TruSeq HT Sample Prep Kits were purchase from Integrated DNA Technologies and used in the ligation step. TruSeq-1 and TruSeq-2 primer were used for PCR enrichment of adaptor ligated DNA. Library size was verified with a Bioanalyzer using an Agilent High Sensitivity DNA kit. The concentration of each library was determined using the KAPA Library Quantification Kit for Illumina platforms. Sequencing was performed by the NIDDK Genomics Core facility using a MiSeq system with the MiSeq 2 x 250 bp Sequencing Kit (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Quality trimming (max ambiguity =2) and trimming of 15 nucleotides from the 5’ terminal of all sequences was performed with CLC Genomics Workbench.
Trimmed data was aligned to the E. coli reference genome (NC_000913.3) and an unmapped reads file was generated.
Differential expression experiments consisted of 3 replicates of MG1655 WT and RpoD D445V mutant E. coli grown in LB at either 37o or 23oC.
Count data was normalized to total reads in CLC Genomics Workbench
Statistical analysis was performed in CLC Genomics Workbench using the EDGE test tagwise dispresions with False Discovery Rate (FDR) corrected p values.
Assembly: NC_000913.3
Supplementary files format and content: Comma seperated value text files include: 1) counts for each gene in all replicates prior to normailzation; 2) normalized values for each gene in all replicates; 3) the fold change of each gene in the two samples as determined in CLC Genomics Workbench using the Baggerley’s Test; 3) False Discovery Rate (FDR) corrected p values.
|
| |
|
| Submission date |
Jan 05, 2023 |
| Last update date |
Jan 10, 2023 |
| Contact name |
Melissa Arroyo-Mendoza |
| E-mail(s) |
[email protected]
|
| Organization name |
NIDDK
|
| Department |
LBG
|
| Lab |
GERS
|
| Street address |
8 CENTER DR RM 2A11
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL17439 |
| Series (1) |
| GSE222248 |
The E. coli pathobiont LF82 encodes a unique variant of sigma 70 that results in global gene expression changes and altered phenotypes |
|
| Relations |
| BioSample |
SAMN32602464 |
| SRA |
SRX18945506 |
