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Sample GSM7133579

Query DataSets for GSM7133579

Status

Public on Apr 03, 2023

Title

Chlor rep 2 time 4

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 100µg/mL rifampicin and placed immediately in RNAprotect bacterial reagent (QIAGEN) at the indicated timepoint (0,1,2,4,8,and 15 min). 0 minute time-points were collected just prior to rifampicin addition.

Growth protocol

WT(Caulobacter crescentus NA1000) 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 ribozero gram negative kit 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 03, 2023

Contact name

Jared Michael Schrader

E-mail(s)

[email protected]

Organization name

Wayne State University

Department

Biological Sciences