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Sample GSM3133356

Query DataSets for GSM3133356

Status

Public on Nov 13, 2018

Title

MumR+H2O2 replicate 3

Sample type

SRA

Source name

Bacterial culture

Organism

Acinetobacter baumannii

Characteristics

strain: ATCC 17978
growth condition: mid-exponential growth in LB; 10 minute exposure to 5 mM H2O2
genotype: mumR-/- strain

Treatment protocol

Cultures were removed from the incubator and treated with 5.65 microliters of 30% H2O2 (EMD Millipore, Darmstadt, DE), for a final concentration of 5 mM, 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, Center Valley, PA) and ethanol (Sigma, St. Louis, MO) and frozen at -70C.

Growth protocol

Acinetobacter baumannii 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 37C with shaking at 180 rpm. Overnight cultures were diluted 1:1000 into 10 mL LB in a 50 mL conical tube and grown to mid-exponential phase.

Extracted molecule

total RNA

Extraction protocol

Cultures were thawed and centrifuged at 7,000 rpm for 10 minutes, supernatants were decanted, and pellets were dried on paper towels. Pellets were resuspended in LETS buffer (0.1 M LiCl, 10 mM EDTA, 10 mM Tris HCl pH 7.4, 1% SDS), homogenized in a bead beater (Fastprep-24, MP Biomedical, Santa Ana, CA) with Lysing Matrix B beads (MP Biomedicals, MP Biomedical, Santa Ana, CA) at a speed of 6 m/s for 45 seconds, heated at 55 °C for 5 minutes, and centrifuged for 10 minutes at 15,000 rpm. The upper phase was collected, mixed with 1 mL TRI Reagent (Sigma, St. Louis, MO), and incubated for 5 minutes at room temperature. Chloroform (0.2 mL, Acros Organics, Waltham, MA) was added, samples were vigorously shaken for 15 seconds, and samples were incubated at room temperature for 2 minutes. Following centrifugation at 4 °C for 15 minutes, 600 mL of the upper aqueous phase was collected and RNA was precipitated with 1 mL isopropanol (Sigma, St. Louis, MO). RNA was washed with 70% ethanol (Sigma, St. Louis, MO) and resuspended in 100 mL DNase-Free, RNase-Free water (ThermoFisher, Waltham, MA). DNA contamination was removed by treatment with 8 mL RQ1 enzyme (Promega, Madison, WI), 12 mL 10X RQ1 buffer, and 2 mL Ribolock RNase inhibitor (ThermoFisher, Waltham, MA) for 2 hours at 37 °C. DNase was removed and samples further purified by the RNEasy kit (Qiagen, Hilden, DE) using the manufacturer’s instructions and RNA was stored long-term at -80 °C.
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

Normalized_expression_4615-JPZ-0010-0027_GEOupload

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 Acinetobacter baumannii ATCC17978.
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

May 07, 2018

Last update date

Nov 13, 2018

Contact name

Lillian Juttukonda

E-mail(s)

lillian.juttukonda@vanderbilt.edu

Organization name

Vanderbilt University

Department

Pathology, Microbiology and Immunology