GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3091613

Query DataSets for GSM3091613

Status

Public on Jul 12, 2018

Title

R4Delta983PL Rep1

Sample type

SRA

Source name

bacterial cellular RNA

Organism

Enterococcus faecalis

Characteristics

strain/genotype: delta-ahrC
sample type: Planktonic (PL)

Growth protocol

E. faecalis biofilm and planktonic cells were obtained from CDC Biofilm Reactors (CBR) that were set-up as previously described (Dale et al., npj Biofilms Microbiomes, 2017). Briefly, OG1RF and ΔahrC were streaked on BHI plates containing rifampicin and fusidic acid. For each reactor, an overnight M9-YE culture was inoculated with three colonies and grown at 37°C. A CBR containing full strength M9-YE was inoculated with 2 ml of an overnight culture at a cell density of 1.4 x 109-2.8 x 109 and incubated with stirring at 37°C for 4 h to allow for bacterial attachment. M9-YE (10%) was then pumped through reactor for additional 2 h. Next, biofilm cells from 24 polycarbonate coupons (BioSurface Technologies Corp., Bozeman, MT) and eight Aclar fluoropolymer strips (Electron Microscopy Sciences, Hatfield, PA) from the reactors were washed, treated with RNAprotect (Qiagen) and vortexed as described in Dale et al, (2017). Three milliliters of planktonic cells were treated with RNAprotect, pelleted and stored with the biofilm cells at -80°C. Two biological replicates were run for each strain. To recover sufficient RNA from ΔahrC, two reactors were pooled for each replicate. For one of the OG1RF replicates, two reactors were also pooled.
Planktonic cells from OG1RF and ΔahrC were also acquired from liquid cultures grown in BHI. Overnight cultures were diluted 1:100 in BHI medium and grown under static conditions until optical density 600 nm reached 0.600-0.650 at 37°C. Three milliliters of cells from each culture were treated with RNAprotect (Qiagen) according to the manufacturer’s protocol. Cells were pelleted and stored at -80°C. Two biological replicates were run for each strain.

Extracted molecule

total RNA

Extraction protocol

Planktonic and biofilm cell pellets, were thawed and resuspended in 200 µl Tris/EDTA/ mutanolysin/lysozyme solution (10 mM Tris [pH 8.0], 1.0 mM EDTA [pH 8.0], mutanolysin (500 U/ml), lysozyme (30 mg/ml)) and incubated at 37⁰C for 10 minutes. To the resuspensions, 700 µl of Buffer RLT (Qiagen RNeasy Mini Kit) with 10 µl β-ME was added and vortexed to mix. RNA was then purified using the Qiagen RNeasy Mini Kit according to the manufacturer’s instructions. Total RNA from biological replicates were treated with TURBO DNase (Ambion /ThermoScientific, Waltham, MA) using the rigorous protocol according to the manufacturer’s instructions. DNase was removed from treated samples using the RNA Clean & Concentrator-25 (Zymo Research). mRNA was enriched by rRNA removal using the MICROBExpress Bacterial mRNA Purification Kit (Ambion/ThermoScientific) according to the manufacturer’s instructions.
University of Minnesota Genomic Center (UMGC) performed QC and library preparation. mRNA quantified using fluorimetry (RiboGreen assay) and RNA integrity assessed using capillary electrophoresis (e.g., Agilent BioAnalyzer 2100), generating an RNA Integrity Number (RIN). For a sample to pass QC, mass must be ≥ 1 ug, with a RIN > 8. Library created using TruSeq RNA V2 kit. Briefly, cDNA was generated using reverse transcriptase, fragmented and blunt-ended, and ligated to indexed (barcoded) adapters. The RNA library was gel size selected to generate inserts of ~200 bp.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Mutant
Growth conditions: M9-YE, CBR
Galaxy33, OG1RF R6_R7_D983_R4_R5 PLvsBIO

Data processing

Illumina RNAseq library creation and sequencing, using paired-end HiSeq 2500 (50 bp and 125 bp), HiSeq (100 bp) and MiSeqv3 (75 bp) platform, were performed at the University of Minnesota Genomics Center (UMGC). The fastq CASAVA (version 1.8 Illumina) sequence files were de-multiplexed and deposited in Minnesota Supercomputer Institute (MSI, University of Minnesota).
Sequence analysis on paired end reads were carried out using Galaxy maintained by MSI. The fastq CASAVA 1.8 files were converted to CASAVA 1.7 and assessed for quality using FastQC Read and Quality Reports. (The libraries run on the HiSeq instrument were run on two lanes (technical replicates) for each condition. The fastq files collected were combined to create a single output file for each condition using the MSI text manipulation tool, concatenate datasets, prior to converting to CASAVA 1.7.)
FASTQ Quality Trimmer by sliding window and cutadapt were used to remove the 3’ends of raw sequencing reads that had a Phred quality score <20 and to remove adapter sequences, respectively.
FASTQ interlacer and FASTQ de-interlacer or resync: Paired-end resynchronization (MSI tool) were used on the trimmed fastqsanger files to remove any unmatched paired end reads followed by mapping to the OG1RF reference genome (NC_017316.1/CP002621.1) using Bowtie for Illumina.
The Bowtie SAM files were sorted by chromosome and start position using Picard Sortsam.
For each condition, Cufflinks was used to assemble the transcripts and determine expression levels (FPKM). The generated gtf files created for biological replicates, for the conditions to be compared, were assembled using Cuffmerge.
Cuffdiff was used to determine significant differences in transcriptional expression between culture conditions with the False Discovery Rate at 0.05.
Genome_build: NC_017316.1/CP002621.1
Supplementary_files_format_and_content: tabular file includes FPKM values, fold change and statistical analysis for Galaxy33, OG1RF R6_R7_D983_R4_R5 PLvsBIO
Supplementary_files_format_and_content: tabular file includes FPKM values, fold change and statistical analysis for Galaxy73, OG1RF vs D983_PL

Submission date

Apr 10, 2018

Last update date

Jul 12, 2018

Contact name

Gary M Dunny

E-mail(s)

dunny001@umn.edu

Organization name

University of Minnesota

Department

Department of Microbiology and Immunology