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Sample GSM2992227

Query DataSets for GSM2992227

Status

Public on Nov 30, 2018

Title

P. involutus T1 1

Sample type

SRA

Source name

Mycelium

Organism

Paxillus involutus

Characteristics

strain: Batsch Fr., ATCC 200175
days: 2
time point: 1
biological replicate: 1

Treatment protocol

After 24 h of nitrogen starvation, the mycelium was washed with sterile water and the SOM extract was added. The extract was supplemented with glucose to a final concentration similar to that in the MMN medium (Rineau et al., 2012). For the production om extract forest litter material was collected from the upper 10-cm soil layer in a 61-year-old Norway spruce stand growing in a N-poor site in central Sweden (soil pH, 5.0) and was extracted using hot water.

Growth protocol

Cultures of P. involutus (Batsch Fr., strain ATCC 200175) and L. bicolor (strain Maire P.D.Orton) were grown in petri dishes on a glass bead layer containing liquid minimum Melin-Norkrans (MMN) medium for 9 and 18 days, respectively. The medium was replaced with MMN medium without N to induce an N-deprived mycelium.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) with the RLC buffer and on-column DNase treatment, according to the manufacturer’s instructions. Total RNA was eluted in H2O.
After reverse transcription into double-stranded cDNA and barcoding by means of the massively parallel signature sequencing protocol, libraries were sequenced (RNA-Seq) using a HiSeq2000 instrument (Illumina Inc.) in single-read mode and with a read length of 50 bp (IGA Technology Services, Italy; www.igatechnology.com).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Active growth

Data processing

FastQC (ver. 0.11.2) (Andrews, 2010) was used for quality control of raw RNA-Seq reads and ribosomal RNA-like sequences were removed using riboPicker (standalone ver. 0.4.3, SILVA database ver. 119) (Schmieder et al., 2012). RNA-Seq reads were aligned to the transcriptome using TopHat2 (ver. 2.0.13) (Kim et al., 2013) with Bowtie2 (ver. 2.2.3.0) (Langmead et al., 2012) and the following parameters: -T -x 1 -I 5000 --library-type 'fr-firststrand' SAMtools (ver. 0.1.19) (Li et al., 2009) was used to sort and convert the BAM files to SAM files. Transcript abundances were measured as read counts in CDS using htseq-count (ver. 0.6.1) (Anders et al., 2015) with the following parameters: -s 'reverse' -t 'CDS' -i 'gene_id' -m 'intersection-nonempty' Genes with mean count below 11 across all samples were removed. Read count normalization factors were calculated for each fungus using EDASeq (ver. 2.8.0) with 50 strata and full quantile normalization to correct for transcript length and G/C-content within each sample (Risso et al., 2011), and combined with sample size factors to adjust for different sequencing depths using DESeq2 (ver. 1.14.1) (Love et al., 2014). Principal component analysis (PCA) was performed on normalized and regularized log-transformed (DESeq2) RNAseq data blinded to the experimental design for quality assurance.
Genome_build: Paxillus involutus ATCC 200175 v1.0 (http://genome.jgi-psf.org/Paxin1/Paxin1.home.html)
Genome_build: Laccaria bicolor v2.0 (http://genome.jgi-psf.org/Lacbi2/Lacbi2.home.html)
Supplementary_files_format_and_content: The tab-delimited Lac_rawCounts.tab and Pax_rawCounts.tab files contain the Joint Genome Institute (JGI) gene model names and raw read counts for each sample.
Supplementary_files_format_and_content: The tab-delimited Lac_normalizationFactors.tab and Pax_normalizationFactors.tab files contain the matrix of normalization factors for each gene and sample. Used in DESeq2 with the raw counts file to correct for gene-specific effects and sample-specific sequencing depths. For correct dispersion estimation the matrix of normalization factors needs first to be scaled to geometric mean near 1 for each row (gene) for correct dispersion estimation (see DESeq2::normalizationFactors).
Supplementary_files_format_and_content: The tab-delimited Lac_rlog_normalizedCounts.tab and Pax_rlog_normalizedCounts.tab files contain the gene model names and DESeq2 regularized log (rlog) transformed normalized counts for each sample.

Submission date

Feb 12, 2018

Last update date

Nov 30, 2018

Contact name

Anders Tunlid

Organization name

Lund University

Department

Biology

Lab

Microbial Ecology