GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3463573

Query DataSets for GSM3463573

Status

Public on Dec 17, 2019

Title

gth__1

Sample type

SRA

Source name

E. coli K12 MG1655

Organism

Escherichia coli

Characteristics

strain: MG1655
media: M9 minimal media w/ 2g/L glucose
supplement: glutathione

Growth protocol

glycerol stocks of E. coli strains were inoculated into M9 minimal media with sample-specific carbon source, supplemented with 1 ml trace element solution (100X). The culture was incubated at 37C overnight with agitation, and then was used to inoculate the fresh media. The fresh culture was incubated at 37C with agitation to the mid-log phase (OD600 ≈ 0.5). For nitrate respiration cultures, a 35:50 ratio of carbon dioxide to nitrogen was bubbled through hte media to deoxygenate.

Extracted molecule

total RNA

Extraction protocol

After inoculation and growth, 3 mL of cell broth (OD600 ~ 0.5) was immediately added to 2 volumes Qiagen RNA-protect Bacteria Reagent (6 mL), vortexed for 5 seconds, incubated at room temperature for 5 min, and immediately centrifuged for 10 min at 17,500 RPMs. The supernatant was decanted, and the cell pellet was stored in the -80°C. Cell pellets were thawed and incubated with Readylyse Lysozyme, SuperaseIn, Protease K, and 20% SDS for 20 minutes at 37°C. Total RNA was isolated and purified using the Qiagen RNeasy Mini Kit columns and following vendor procedures. An on-column DNase-treatment was performed for 30 minutes at room temperature. RNA was quantified using a Nano drop and quality assessed by running an RNA-nano chip on a bioanalyzer. The rRNA was removed using Illumina Ribo-Zero rRNA removal kit for Gram Negative Bacteria. A KAPA Stranded RNA-Seq Kit (Kapa Biosystems KK8401) was used following the manufacturer’s protocol to create sequencing libraries with an average insert length of around ~300 bp.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Raw sequencing reads were mapped to the reference genome (NC_000913.3) using bowtie v1.1.2 with parameters -X 1000 -n 2
Transcript abundance was quantified using summarizeOverlaps from the R GenomicAlignments package, with strand inversion for the dUTP protocol and “IntersectionStrict” mode
Transcripts per Million (TPM) were calculated using DESeq2’s fpkm function, then normalizing for library size.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: Comma-separated text files include TPM (Transcripts per million) for each sample

Submission date

Nov 07, 2018

Last update date

Dec 17, 2019

Contact name

Anand Sastry

E-mail(s)

[email protected]

Organization name

University of California, San Diego

Department

Bioengineering