 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 03, 2023 |
| Title |
WT 9 min rep 1 |
| Sample type |
SRA |
| |
|
| Source name |
NA1000
|
| Organism |
Caulobacter vibrioides |
| Characteristics |
cell line: NA1000
cell type: C. crescentus cells
genotype: WT
|
| Treatment protocol |
The cells were treated with 200µg/mL rifampicin and placed immediately in RNAprotect bacterial reagent (QIAGEN) at the indicated timepoint (0,1,3,9 min). 0 minute time-points were collected just prior to rifampicin addition.
|
| Growth protocol |
WT(Caulobacter crescentus NA1000) cells, delta rhlB(∆rhlB) and delta rppH (∆rppH cells) were grown in M2G media overnight at 28 C. The next day, the cells were diluted and grown until midlog-phase (0.3 to 0.5 OD600) which were used for mRNA half-life profiling.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were pelleted at 20,000 x g for 1 min in 1.5 mL eppendorf tubes to prepare for RNA extraction.Pelleted cells were resuspended in 1mL of pre-warmed (to 65 0C) TRizol Reagent (Ambion) and incubated for 10 min at 65 0C in a heat block. 200 mL of chloroform were added to the samples and tubes were gently inverted a few times. Then samples were incubated at room temperature for 5 min and spun at 20,000 x g for 10 min. The supernatant was moved to siliconized tubes and 500 mL of chloroform was added, vortexed, and spun at 20,000 x g for 10 minutes. RNA samples were precipitated using isopropanol (1x volume isopropanol, 0.1X volume 5M NaOAc pH 5.2) overnight at – 80 0C. The RNA samples were spun at 20,000 x g at 4 0C for 1 hour. Pellets were washed with cold 80% ethanol and spun for 10 min at 20,000 x g, air-dried, and resuspended in 10 mM Tris-HCl (pH 7.0). The RNA-Seq libraries were made using 5µg of total RNA of each sample. Ribosomal RNAs were removed by riboPOOLs (oligos designed for removal of Caulobacter crescentus rRNAs from siTOOLs Biotech) and library construction was performed according to the protocol in (Aretakis et al., 2018).
mRNA fragments were ligated to 5' adenylated DNA oligo. After reverse transcription, the single stranded DNA was circularized, and PCR amplified [Oh et al,. Cell 147, 1295 (2011)]. Updated adapter oligos that are compatible with the novaseq were used from [Aretakis et al. Methods in Enzymology (2019)].
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Single end reads were first stripped of 3' ligated adapter using a custom python script.
Adapter stripped reads were then aligned to rRNA using bowtie 0.12.8 to remove remaining rRNA reads. Reads that did not align to rRNA were then aligned to the full C. crescentus NA1000 genome using bowtie 0.12.8.
mRNA levels were then calcualted by normalizing using the reads per kilobase per million mapped reads (RPKM). The relative mRNA levels over time were then calculated using the rifcorrect software [Aretakis et al. BioRxiv 2022].
Assembly: NA1000 operon annotation (Bharmal et al. MRA 2020)
Supplementary files format and content: tab delimited text
|
| |
|
| Submission date |
Mar 30, 2023 |
| Last update date |
Apr 04, 2023 |
| Contact name |
Jared Michael Schrader |
| E-mail(s) |
[email protected]
|
| Organization name |
Wayne State University
|
| Department |
Biological Sciences
|
| Lab |
Schrader Lab
|
| Street address |
5047 Gullen Mall, Biological Sciences Building Room 2168
|
| City |
Detroit |
| State/province |
MI |
| ZIP/Postal code |
48202 |
| Country |
USA |
| |
|
| Platform ID |
GPL32153 |
| Series (1) |
| GSE228592 |
WT, ∆rhlB and ∆rppH mRNA half-life profiling. |
|
| Relations |
| BioSample |
SAMN34000505 |
| SRA |
SRX19832925 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7133597_WT9L1.txt.gz |
40.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
