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Sample GSM5097905

Query DataSets for GSM5097905

Status

Public on Nov 11, 2022

Title

CA-0M-4

Sample type

SRA

Source name

in vitro mono-species biofilm

Organism

Candida albicans

Characteristics

strain: SC5314
age: 28-hour biofilm
treatment: untreated

Treatment protocol

After 24 h, the MHB media was replaced with MHB ± 5 μg/mL meropenem.

Growth protocol

P.aeruginosa was maintained and cultured in Miller modified LB; C. albicans in YPD. Both were grown at 37°C, with aeration, at 200 rpm overnight. Cultures were washed twice in PBS and P. aeruginosa cultures diluted to OD600 of 0.2, and C. albicans cultures diluted to 1×10^6 cells/mL, in Mueller Hinton broth (MHB). 300 µl of the P. aeruginosa culture and/or 3 mL of the C. albicans culture were added to flat-bottom 6-well plates, and the total volume was made up to 6mL per well with MHB. Single- and dual-species biofilms were incubated in triplicate, statically at 37°C for 24 h.

Extracted molecule

total RNA

Extraction protocol

After 4 h of treatment, the media was removed and replaced with 2 mL PBS + 50 µg/mL DNase, which had been incubated at 37°C. Sterile cell scrapers were used to detach biofilm cells and technical replicates were pooled and mixed in a 15-mL Falcon tube, on ice. The biofilm cell samples were centrifuged at 3500 rpm, at 4°C, for 5 min, to form cell pellets. The supernatant was removed, and cell pellets were snap frozen in liquid nitrogen, before being stored at -70°C. Samples were packaged on dry ice and shipped to GeneWiz®, UK, for RNA extraction, sequencing and analysis. RNA extraction was performed using the QIAGEN Rneasy Plus mini Kit.
1) Ribosomal RNA depletion, 2) RNA fragmentation and random priming, 3) first and second strand cDNA synthesis, end repair, 5' phosphorylation and dA-tailing, 4) adapter ligation, PCR enrichment and sequencing. Sequencing: Illumina HiSeq, 2x150bp configuration, single index, per lane.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Evaluate sequence quality: #reads; yield(Mbases); mean quality score; % bases >=30. Per base sequence quality: FastQC. Per sequence GC content: FastQC.
Mapping sequence reads to the reference genome: Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36.
The trimmed reads were mapped to the C_albicans_SC5314 (http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly21/current/) or the P_aeruginosa_PAO1 (https://www.ncbi.nlm.nih.gov/nuccore/NC_002516.2) reference genomes available on ENSEMBL separately using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step.
Extracting gene hit counts: Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. The hit counts were summarized and reported using the gene_id feature in the annotation file. Only unique reads that fell within exon regions were counted.
Differential expression was calculated using DESeq2
Genome_build: C. albicans SC5314 (http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly21/current/) or P. aeruginosa PAO1 (https://www.ncbi.nlm.nih.gov/nuccore/NC_002516.2)
Supplementary_files_format_and_content: Excel work books containing the DEseq2 results for the multiple comparisons performed during the analysis. Each file contains the normalised reads for each sample, p value, adjusted p value, and the log2 expression value

Submission date

Feb 19, 2021

Last update date

Nov 11, 2022

Contact name

Rebecca Anne Hall

E-mail(s)

r.a.hall@kent.ac.uk

Organization name

University of Kent

Department

School of Biosciences