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Sample GSM2222252

Query DataSets for GSM2222252

Status

Public on Jul 01, 2016

Title

rep 1_12 hpi

Sample type

SRA

Source name

hyphae and area of leaf under hyphae

Organisms

Brassica napus; Sclerotinia sclerotiorum

Characteristics

tissue: hyphae and area of leaf under hyphae
sclerotinia sclerotiorum strain: Sclerotinia sclerotiorum 1980
brassica napus strain: Brassica napus DH12075
time: 12 hours post innoculation

Treatment protocol

One gram of mycelia (wet weight) was spread over a 5-cm diameter circle on a detached B. napus leaf and incubated in a sealed, humidified tray at room temperature. After the appropriate infection time, mycelia and the leaf material under the mycelia were collected and flash frozen in liquid nitrogen.

Growth protocol

The sclerotinia was grown on minimal salts-glucose (MS–Glu: 2 g/L NH4NO3, 1 g/L KH2PO4, 0.1 g/L MgSO4:7H2O, 0.5 g/L yeast extract, 3 g/L DL-malic acid, 1 g/L NaOH, supplemented with 1% glucose) medium at room temperature, shaken continuously at 40 rpm for 72 hours.

Extracted molecule

polyA RNA

Extraction protocol

The samples were ground to a fine powder with an RNAse-free mortar and pestle precooled with liquid nitrogen. Total RNA was extracted using an Illustra RNAspin mini RNA isolation kit (Illumina, San Diego, USA). RNA quantity and quality was assessed using a Qubit fluorometry assay (Invitrogen Corp., Carlsbad, CA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively.
Libraries were prepared using a Truseq stranded mRNA kit (Illumina, San Diego, USA) following the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Data processing

libraries were sequenced on an Illumina MiSeq, running Control Software version 2.5.0.5 using default parameters
Reads were trimmed by CLC genomics workbench 7.0.4. Sequences were trimmed based on quality (limit 0.05) and ambiguity (max of 2 ambiguous bases). MiSeq adaptors were removed from both strands.
RNAseq analysis was performed by CLC genomics workbench 7.0.4. Parameters were: mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.8, similarity fraction 0.8, max of 10 hits for a read
Gene expression was estimated by extracting read counts as integers from the CLC Genomics alignments. The count data were normalized to generate effective library sizes using the scaling method Trimmed Means of M-values (TMM). Statistical analysis was performed with these data using a generalized linear model linked to the negative binomial distribution performed using the EdgeR package (Robinson and Oshlack, 2010). Pair-wise analyses were performed to assess differential gene expression using the control library as a common reference standard.
Genes were considered differentially expressed where the probability after adjustment for multiple hypothesis testing [false discovery rate (FDR)] was less than 0.05. The extent of the observed differential expression was considered meaningful if the fold change exceeded a factor of two.
All significantly up-regulated genes at different sampling times were assigned a functional classification using the BLAST2GO plugin (v1.4.4) in the CLC Genomics Workbench 8.0.1 for functional annotation using Interpro and the NCBI refseq protein database. Then gene ontology (GO) terms to each gene were extracted. The results were filtered to remove top-level annotations and apply the GO-slim categorization from Aspergillus in order to summarize the results. Blast2GO ran ANNEX (Myhre et al., 2006) to add implicit GO terms for a more complete annotation. Finally, Blast2GO was used to calculate the abundance of GO classifications for the significantly up-regulated genes for each time point.
Genome_build: Sclerotinia sclerotiorum 2 transcripts (Broad Institute)
Supplementary_files_format_and_content: MS Excel file containing the counts per million (counts normalized for library size)

Submission date

Jun 30, 2016

Last update date

May 15, 2019

Contact name

Dwayne Hegedus

E-mail(s)

[email protected]

Phone

(306) 385-9427

Organization name

Agriculture and Agri-Food Canada