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Sample GSM3461157

Query DataSets for GSM3461157

Status

Public on Dec 17, 2019

Title

MG_no_te_2

Sample type

SRA

Source name

E. coli K12 MG1655

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

strain: MG1655
media: M9 minimal media w/ 0.2% glucose w/o trace elements
knock-out: --

Growth protocol

glycerol stocks of E. coli strains were inoculated into 0.2%(w/v) glucose M9 minimal media. M9 minimal media was also supplemented with 1 ml trace element solution (100X), excluding MG_no_te_1 and MG_no_te_2. The culture was incubated at 37 ℃ overnight with agitation, and then was used to inoculate the fresh media. The fresh culture was incubated at 37 ℃ with agitation to the mid-log phase (OD600 ≈ 0.5). 10mM glutamate was added to the media MG_glu_1 and MG_glu_2.

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 MiSeq

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 06, 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