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Sample GSM3911394 Query DataSets for GSM3911394
Status Public on Jun 29, 2019
Title JS299 1 min
Sample type SRA
Source name JS299 half-life profiling
Organism Caulobacter vibrioides NA1000
Characteristics strain: NA1000
isolate: Strain JS299
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
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 GPL26862
Series (1)
GSE133532 BR-body mutant mRNA half-life profiling.
BioSample SAMN12163263
SRA SRX6384239

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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