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Sample GSM4226339

Query DataSets for GSM4226339

Status

Public on Mar 31, 2020

Title

WTtargocilreplicate3

Sample type

SRA

Source name

Bacterial culture

Organism

Bacillus anthracis

Characteristics

strain: Sterne
protocol: mid-exponential growth in LB, 10 minute exposure to 10 microgram /mL targocil
genotype: WT parental strain biological replicate 3

Treatment protocol

Cultures were removed from the incubator and treated with 5 microliters of 10 mg/mL targocil (MedChemExpress), for a final concentration of 10 μg/mL, or opened but not treated. After 10 minutes of incubation at 37C with shaking at 180 rpm, cultures were immediately mixed with 10 mL of a 1:1 mixture of ice-cold acetone (JT Baker) and ethanol (Sigma) and frozen at -80C.

Growth protocol

Bacillus anthracis cultures were prepared using colonies streaked to isolation from frozen stocks 2 days prior to the experiment. Overnight cultures in biological triplicate were started from a single colony and grown for sixteen hours in luria broth at 30C with shaking at 180 rpm. Overnight cultures were diluted 1:100 into 3 mL LB in a 15 mL conical tube and grown to mid-exponential phase (6 hr)

Extracted molecule

total RNA

Extraction protocol

Cultures were thawed and centrifuged at 7,000 rpm for 10 minutes, supernatants were decanted, RNA was isolated using the RNeasy kit (Qiagen) and DNA was removed using the Turbo DNA-free kit (Invitrogen, Thermo Scientific)
Five hundred ng of total RNA was required for proceeding to downstream RNA-seq applications. First, ribosomal RNA (rRNA) was removed using Ribo-Zero™ Gold (Epidemiology) kit (Illumina, San Diego, CA) using manufacturer's recommended protocol. Immediately after the rRNA removal the RNA was fragmented and primed for the first strand synthesis using the NEBnext First Strand synthesis module (New England BioLabs Inc., Ipswich, MA). Directional second strand synthesis was performed using NEBNExt Ultra Directional second strand synthesis kit. Following this the samples were taken into standard library preparation protocol using NEBNext® DNA Library Prep Master Mix Set for Illumina® with slight modifications. Briefly, end-repair was done followed by polyA addition and custom adapter ligation. Post-ligated materials were individually barcoded with unique in-house Genomic Services Lab (GSL) primers and amplified through 12 cycles of PCR. Library quantity was assessed by Qubit 2.0 Fluorometer, and the library quality was estimated by utilizing a DNA High Sense chip on an Caliper Gx (Perkin Elmer). Accurate quantification of the final libraries for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA). Each library was diluted to a final concentration of 12.5nM and pooled equimolar prior to clustering.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

mid-exponential growth in LB, 10 minute exposure to 10 μg/mL targocil

Data processing

Quality control checks on raw sequence data from each sample were performed using FastQC (Babraham Bioinformatics, London, UK).
Alignment: Raw reads were imported onto the commercial data analysis platform, Avadis NGS (Strand Scientifics, CA, USA) and mapped to the reference Bacillus anthracis Sterne (NCBI Reference Sequence: NC_005945.1)
Filtering: After quality inspection, the aligned reads were filtered on the basis of read quality metrics where reads with a base quality score less than 30, alignment score less than 95, and mapping quality less than 40 were removed. Remaining reads were then filtered on the basis of their read statistics, where missing mates, translocated, unaligned and flipped reads were removed. The reads list was then filtered to remove duplicates.
Generalization of normalized abundance measurements: Samples were grouped and quantification of transcript abundance was done on this final read list using Trimmed Means of M-values (TMM) (Robinson and Oshlack 2010) as the normalization method. Reference: Robinson MD, Oshlack A. 2010. A scaling normalization method for differential expression analysis of RNA-seq data. Genome biology 11:R25.
Supplementary_files_format_and_content: Comma separate values file containing gene symbol, chromosome, gene start and end, strand, and log2 normalized expression by sample

Submission date

Dec 19, 2019

Last update date

Mar 31, 2020

Contact name

Clare Laut

E-mail(s)

clare.l.laut@vanderbilt.edu

Organization name

Vanderbilt University

Street address

1161 21st Avenue South Medical Center North RM# A5104