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Sample GSM2516627

Query DataSets for GSM2516627

Status

Public on Feb 20, 2018

Title

9B_MG+PMA_t30

Sample type

SRA

Source name

E. coli

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

treatment: MG1655 + 3µM PMA at t30

Treatment protocol

When cultures reached OD595 ≈ 0.470 (~ 200 min), two cultures were made 3 µM mercuric chloride (HgCl2) or 3 µM phenylmercuric-acetate (PMA) and the third was left as an unexposed control. Duplicate 1 ml aliquots of each culture were collected at 0 (unexposed control only), 10, 30, 60 min after mercurial exposure and immediately centrifuged at 21 krpm, for 3 min at 4°C. Spent medium was aspirated and cell pellets were frozen at -70°C within 5 min after collection.

Growth protocol

For each biological RNA-Seq replicate E coli K-12 MG1655 was subcultured from cyrostorage on Luria-Bertani (LB) agar overnight at 37°C. A half-dozen well-isolated colonies were used to inoculate a 20 ml starter culture in Neidhardt MOPS Minimal Medium (NM3) (Neidhardt et al., 1974, J Bacteriol) (0.2% final glucose concentration) supplemented with 20 mg/L uracil and 500 µg/L thiamine, which was incubated at 37°C with shaking at 250 rpm overnight (~18 hr). The overnight starter culture was diluted 1:30 to initiate the experimental culture and divided into three 500 ml flasks with 100 ml NM3 in each, which were incubated at 37°C with shaking at 250 rpm.

Extracted molecule

total RNA

Extraction protocol

One cell pellet from each condition and sampling time was thawed on ice; total RNA was isolated by RNAsnap™ (Stead et al., 2012, Nucleic Acids Res). DNA contamination was removed by two treatments with Turbo-DNase (Ambion; Life Technologies). Ribosomal RNA depletion was performed with the Ribo-Zero™ rRNA removal kit for Gram-negative bacteria (Epicentre) and concentrated using RNA Clean and Concentrator™ -5 columns (Zymo Research) following the manufacturer’s instructions.
The quality and quantity of rRNA-depleted RNA was assessed on a 2100 Bioanalyzer RNA pico chip (Agilent Technologies) using manufacturer’s recommendations. Next generation sequencing (NGS) libraries were prepared using the Kapa biosystems NGS stranded library prep kit for RNA-Seq with dual indexed Illumina adapters. The mode library insert size was ~150 bp, as determined by high sensitivity NGS fragment analysis kit for Fragment Analyzer™ (Advanced Analytical Technologies) using manufacturer’s instructions. Quantification of each library was done by qPCR and all 30 libraries were pooled in equal concentrations.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

rRNA-depleted total RNA
MG1655 + 3µM PMA at t30 (biological rep. 2)

Data processing

Quality control processing of sequence data was performed using Galaxy (https://galaxyproject.org). The FASTX tools in Galaxy (http://hannonlab.cshl.edu/fastx_toolkit) were used for filtering by quality (80% of sequence > quality score of 20), then reads were trimmed at both 5’ and 3’ ends using a window and step size of 1 with quality score > 20.
Forward- and reverse-read mate-pairs were assembled and aligned to the Escherichia coli MG1655 K-12 genome using Bowtie2 (Langmead and Salzberg, 2012, Nat Methods). SAMtools (Li et al., 2009, Bioinformatics) was used to convert Bowtie2 output (.bam file) to SAM format.
The number of sequence reads that aligned to features in annotation file (Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.2.24.gtf from http://bacteria.ensembl.org) were tabulated from the resulting SAM alignment files using the HTSeq-count program (Anders et al., 2015, Bioinformatics) with intersection non-empty mode.
Mapped read counts were analyzed for differential expression (false discovery rate of < 0.01, fold-change > 2) using the baySeq package in R (Hardcastle and Kelly, 2010, BMC Bioinformatics). Within baySeq, two-way comparisons using quantile normalization were made for all three biological replicate transcriptomes over time for HgCl2 exposure or PMA exposure versus the unexposed control.
Genome_build: ASM584v2 (Escherichia_coli_str_k_12_substr_mg1655.GCA_000005845.2.24.gtf )
Supplementary_files_format_and_content: raw read counts as determined by HTseq-Count, provided in .csv format

Submission date

Mar 01, 2017

Last update date

May 15, 2019

Contact name

Stephen Patrick LaVoie

E-mail(s)

slavoie5@gmail.com

Phone

706-542-2664

Organization name

University of Georgia