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Sample GSM2123144

Query DataSets for GSM2123144

Status

Public on Jul 22, 2016

Title

Third replicate of four species biofilm

Sample type

SRA

Source name

four species biofilm

Organisms

Paenibacillus amylolyticus; Microbacterium oxydans; Stenotrophomonas rhizophila; Xanthomonas retroflexus

Characteristics

biofilm type: Four species

Treatment protocol

A total of 4ml of single-species, dual-species or four-species diluted cultures (mixtures of each species at equal cell density) were inoculated in six-well polystyrene plates (Greiner Bio-One, Frickenhausen, Germany) and allowed to grow for 24h at 24°C.

Growth protocol

Over night cultures of each strain were subcultured to exponential phase and adjusted to an optical density at 600nm (OD600nm) of 0.15 in fresh tryptic soy broth

Extracted molecule

total RNA

Extraction protocol

For RNA extraction, planktonic cells from biofilm-containing wells were gently removed, wells were rinsed three times using phosphate buffer saline (PBS) and then the biofilms were scraped with sterile pipette tips into 1ml of PBS. The biofilm suspensions were immediately centrifuged at 5000g for 5 min and resuspended in 100μl of PBS followed by addition of 500μl Ambion RNAlater (Thermo Fisher Scientific, Waltham, MA, USA). The RNAlater-preserved samples were kept at 4°C ON, after which RNAlater was removed by adding 600μl cold PBS and centrifuged at 8000g for 5 min at 4°C. The pellet was resuspended in 200μl lysozyme solution (20μg/ml) and incubated at room temperature for 10 min with vortexing for 10s every 2 min. After adding 700μl RLT buffer and vortexing for 10s, the obtained solution was transferred into bead-beating tubes (provided by the FastDNA SPIN Kit for soil) and bead beaten using the Savant FastPrep FP120 (Qbiogene) for 30s at setting 5.0, followed by centrifugation at 13000g for 8 min at 4°C. 850μl of supernatant were transferred to a new microcentrifuge tube. 470μl of 100% ethanol was added and mixed by pipetting. 700μl lysate was transferred to an RNeasy mini spin column. Total RNA was then further purified from each biofilm sample using the RNeasy mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. DNase treatment to remove genomic DNA was conducted according to the instructions in Ambion DNAfree Kit (Thermo Fisher Scientific), except the incubation time was extended to 1h. The complete removal of DNA was confirmed by RT-qPCR using 20µl SYBR Green reactions on Mx 3000 (Stratagene, Cedar Creek, Texas). The qPCR targeted the 16S rRNA gene with the eubacterial primers EUB338 and EUB518. All qPCR reactions were run in technical duplicates and contained 10µl Brilliant III SYBR Green QPCR Master Mix (Stratagene, Cedar Creek, Texas), 1µl forward primer (final concentration 385 nM), 1µl reverse primer (final concentration 385 nM), 1µl template DNA (100x diluted to avoid PCR inhibitors. The program combined the annealing and extension step: 95°C for 3 min followed by 40 cycles of 95°C for 10s, 60°C for 20s, and a final dissociation curve. The standard curve for bacteria was made from a pure culture of Pseudomonas putida kt2440 (Park and Crowley, 2005; VanGuilder et al., 2008). mRNA was subsequently isolated using the Ambion MicroExpress Bacterial mRNA Purification Kit (Thermo Fisher) following the manufacturer’s instructions.
The RNA transcripts were prepared using ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre Biotechnologies, Madison, WI, USA)
50bp single reads on an Illumina HiSeq 2000 (Illumina)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

A_3

Data processing

As a quality control, only reads approved by the CASAVA v1.8.2 (Illumina, San Diego, CA, USA) was included and adaptor remnants were removed. End nucleotides with a quality Phred score below 20 were removed. After the end trim process, sequences shorter than 45 nt were removed from the metatranscriptomic libraries. Reads were discarded if they displayed a mean Phred score below 15 in a sliding window of 5 nt for the metatranscriptomes. The quality filtering was carried out using Biopieces (https://github.com/maasha/biopieces).
To create a mapping template for the transcriptomes all four genomes were sequences and assemblied. All metatranscriptome libraries were mapped to the four genomes using Bowtie2 v.2.0.5
An expression table was created by counting number of nt mapping to each gene in the gene catalogue for every sample.
Genome_build: Custom genome catalogue
Supplementary_files_format_and_content: There are two processed data files: custom genome catalogue (all_contigs500.fasta - fasta file format) and a gene expression matrix (matrix_sum.txt - tab separated table in text format).Only reads approved by the CASAVA v1.8.2 (Illumina, San Diego, CA, USA) was included and adaptor remnants were removed. End nucleotides with a quality Phred score below 20 were removed. After the end trim process, sequences shorter than 50 nt were removed from the genomic. Reads were discarded if they displayed a mean Phred score below 10 in a sliding window of 10 nt for the genomic libraries. The quality filtering was carried out using Biopieces (https://github.com/maasha/biopieces). Velvet v.1.2.07 was used to assemble the genomic data (Zerbino and Birney, 2008) and Prodigal v2.50 was used for gene calling (Hyatt et al., 2010) and all contigs above 500 nt was concatenated into a custom genome catalogue. The transcripts were mapped to this catalogue and the information from the gene calling about the position of the genes were used to count how many nt mapped to a specific gene. This information was collected in a gene expression matrix.
Supplementary_files_format_and_content: Please note that the 'prodigal_all_new.txt' contains gene names (which are listed in the matrix_sum.txt) and their position in the genomes, which are included in the file 'all_contigs_500.fasta'.

Submission date

Apr 14, 2016

Last update date

May 15, 2019

Contact name

Lea Benedicte Skov Hansen

E-mail(s)

[email protected]

Organization name

Technical University of Denmark

Department

Center for Biological Sequence Analysis