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Sample GSM1582804

Query DataSets for GSM1582804

Status

Public on Sep 24, 2015

Title

L. bicolor MMN 2

Sample type

SRA

Source name

Mycelium_Laccaria bicolor

Organism

Laccaria bicolor

Characteristics

tissue: Mycelium
strain: (Maire) P.D.Orton
substrate: MMN
barcode: GGCTAC
genome build: Laccaria bicolor v2.0 (http://genome.jgi-psf.org/Lacbi2/Lacbi2.home.html)

Treatment protocol

The fungi were grown on either a minimum Melin-Norkrans medium (MMN) as a reference or a forest-hot litter extract (FH).

Growth protocol

Nine fungal species were analyzed including five ECM and four BR fungi. The fungi were grown in Petri dishes on a layer of glass beads immersed in MMN medium. After 9 days of incubation (18 °C and in the dark), the MMN medium was removed with a sterile pipette. The glass beads and the mycelia were washed with 10 ml of sterile MilliQ (MQ) water, and 10 ml of MMN medium without N was added to induce an N-deprived mycelium. After 24 h the mycelium was again washed in MQ water, and finally the organic matter extracts (10 ml) were added. The organic matter extracts were supplemented with glucose to a final concentration similar to that in the MMN medium. The fungi were grown in either the MMN or the organic matter medium using three replicates, respectively. The cultures were incubated for seven days at 18 °C in the dark.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) with the RLC buffer and the on-column DNase treatment according to the manufacturer. Total RNA was eluted in H2O and stored at -20°C until use. For quality assessments all samples were inspected using a RNA 6000 Nano kit on a 2100 Bioanalyzer (Agilent).
TruSeq mRNA-seq sample prep kit (Illumina)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

processed_data file = LAB_DE.tab

Data processing

Genome sequences and annotations files in GFF-format were downloaded from the Joint Genome Insitute, JGI, at http://genome.jgi.doe.gov/. The format of these files are not accepted by the mapping software TopHat2 2.0.8b [1]. A custom made script was run to reformat the files (available on request).
TopHat2 was run with the settings: -T -x 1 -I 5000
The output file in BAM-format was sorted on positions with SAMtools 0.1.19-44428cd [2].
To count the number of reads mapping to genes the software htseq-count 0.5.4p3 [3] was run with the following parameters: -m intersection-nonempty -i gene_id -t CDS
The R package DESeq 1.10.1 [4] was used to normalize the raw read counts of each species and to identify differentially expressed genes, between FH and MMN, with a Benjamini-Hochberg adjusted p-value of < 0.01.
The Proteinortho 4.26 tool [5] was used with default settings to identify one-to-one orthologs in all nine species. The raw read counts of the orthologs were normalized using DESeq 1.14.0.
References:
[1] Kim D, et al. (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14(4):R36.
[2] Li H, et al.; 1000 Genome Project Data Processing Subgroup (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16):2078–2079.
[3] Anders S, Pyl PT, Huber W (2014) HTSeq – a Python framework to work with high-throughput sequencing data. Bioinformatics. doi:10.1093/bioinformatics/btu638
[4] Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11(10):R106.
[5] Lechner M, et al. (2011) Proteinortho: detection of (Co-)orthologs in large-scale analysis. BMC Bioinformatics 12(1):124.
Supplementary_files_format_and_content: The tar archive "all_species_DE.tab.tar.gz" contains the tab-delimited files "*_DE.tab" with the following columns: Joint Genome Institute (JGI) gene model names, JGI protein IDs, normalized read counts for each of the replicates, fold change values between FH versus MMN, p values and p values adjusted for multiple testing with the Benjamini-Hochberg procedure; The tab-delimited file "Orthologs_Expression_Values.tab" contains ortholog IDs and the normalized expression values of the proteins in each ortholog group; The tab-delimited file "Orthologs_Protein_IDs.tab" contains ortholog IDs and the JGI protein IDs of the nine species in each ortholog group.

Submission date

Jan 12, 2015

Last update date

May 15, 2019

Contact name

Anders Tunlid

Organization name

Lund University

Department

Biology

Lab

Microbial Ecology