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Sample GSM3911389

Query DataSets for GSM3911389

Status

Public on Jun 29, 2019

Title

JS233 0 min

Sample type

SRA

Source name

JS233 half-life profiling

Organism

Caulobacter vibrioides NA1000

Characteristics

strain: NA1000
isolate: Strain JS233

Treatment protocol

The cells were innoculated with 200ug/mL rifampicin and placed immediately in RNAprotect bacterial reagent (qiagen) at the indicated timepoint. 0 min time-points were collected just prior to rifampicin addition.

Growth protocol

5mL of JS299 cells were grown overnight in PYE medium supplied with 0.2% xylose, 0.5µg/mL gentamycin, and 5µg/mL kanamycin. The overnight cultures were then washed 3 times with PYE growth media and used to inoculate 30 mL of PYE medium supplied with 500µM vanillate, 0.5µg/mL gentamycin, and 5µg/mL kanamycin. The cells were grown for 8 hours and then before rif-treatment.

Extracted molecule

total RNA

Extraction protocol

The cells were pelleted at (20,000xg) for 1 min and resuspended in 1mL of 65 °C hot TRizol Reagent (Ambion) and incubated 65 °C for 10 min in a heat block. 200µL of chloroform were added to the samples and the tubes were incubated at room temperature for 5 min before spinning at (20,000xg) for 10 min. RNA samples were chloroform extracted once and precipitated using isopropanol (1x volume isopropanol, 0.1X volume 5M NaOAc pH 5.2) overnight at −80 °C. The RNA samples were spun at 20,000 x g at 4 °C for 1 hour, pellets were washed with 80% ethanol for 10 min, air dried, and resuspended in 10 mM Tris-HCl (pH 7.0). The RNA-Seq libraries were made using 5µg of total RNA samples, rRNA was removed by ribozero gram negative kit (illumina), and library construction was performed according to protocol (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)].

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Reads were mapped to the Caulobacter NA1000 genome (CP001340) using bowtie 0.12.8.
For each aligned read >=20 bases, the 5' nucleotide was used to measure fragment counts within each transcript. For transcripts with >25 fragments at a given timepoint, the RPKM was calculated.
Genome_build: CP001340
Supplementary_files_format_and_content: Each line in the processed file is for a different transcript and contains many columns: Col 1: operon/locus ID. Col 2.-on: strain and indicated time point RPKM value.

Submission date

Jun 28, 2019

Last update date

Jul 07, 2019

Contact name

Jared Michael Schrader

E-mail(s)

schrader@wayne.edu

Organization name

Wayne State University

Department

Biological Sciences