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Sample GSM3350367

Query DataSets for GSM3350367

Status

Public on Aug 23, 2019

Title

P11-2

Sample type

SRA

Source name

mycelia

Organism

Aspergillus niger

Characteristics

cell type: mycelia
substrate: glucose
plate: B
compartment: 2

Growth protocol

A newly developed culturing system called the pie-plate was used that is a variation on the previously described ring-plate (Levin, A.M., de Vries, R.P. & Wosten, H.A. Localization of protein secretion in fungal colonies using a novel culturing technique; the ring-plate system. Journal of microbiological methods 69, 399-401 (2007). The pie-plate with liquid medium (no agar) was covered by a perforated polycarbonate membrane that allows the transfer of proteins and metabolites, but not of fungal hyphae. The fungus was inoculated at the center of the plate and grew radially. The carbon source present in the compartments influenced the radial growth, resulting in a non-symmetrical colony. The compartments were physically separated and therefore influence of one compartment to the other has to be mediated by the fungal mycelium. To evaluate influence of one compartment on the other, the same carbon source was used in three neighboring compartments. This means that one compartment was surrounded by compartments with the same carbon source, while the other two had a different carbon source on one side.

Extracted molecule

total RNA

Extraction protocol

The mycelia growing on each of the compartments was sampled by cutting through the polycarbonate (PC) membrane along the boundary of the compartment with a scalpel blade and then the PC membrane was peeled away before transferring the mycelia to a 2 mL tube and flash freezing in liquid nitrogen; these samples were used for RNA isolation as described previously (PMID:26314379).
The SMARTerR UltraTM Low RNA kit (catalog number 634851) was used to generate full length cDNA template library according to the instruction manual, followed by Ion Plus fragment library kit (Thermo Fisher Scientific, cat# 4471252) to fragment cDNA and barcode adapter ligation for template prep. Template library was quantified using the Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific, cat# 4468802). The emulsion clonal bead amplification to generate bead templates for Ion Torrent platform using the Ion PI™ Hi-Q™ Chef Kit (Thermo Fisher Scientific, A27198) with Ion PI™ Chip Kit v3 (Thermo Fisher Scientific, cat#A26771) on the Ion Chef System (Thermo Fisher Scientific). Sequencing was performed using the Ion PI™ Hi-Q™ Sequencing 200 Kit (Thermo Fisher Scientific, cat# A26772) on Ion Proton sequencer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent Proton

Data processing

Data quality was assessed with htseq-qa
Reads were aligned to the Aspergillus niger NRRL3 genome (https://genome.jgi.doe.gov/portal/pages/dynamicOrganismDownload.jsf?organism=Aspni_NRRL3_1) using TMAP 3.0.1 (https://github.com/iontorrent/TS/tree/master/Analysis/TMAP) , the ION Torrent aligner designed for ION data, with the –g parameter set to 0 (soft clip right and left ends of each read) and the –a parameter set to 1 (return only best alignment, random choice in case of ties).
BAM file outputs were then mapped to genes using htseq-count (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287950/ ) using default settings.
Differential expression analysis was conducted using the R package DESeq2 (Love MI, Huber W, Anders S (2014). “Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.” Genome Biology, 15, 550. doi: 10.1186/s13059-014-0550-8).
Genome_build: NRRL3
Supplementary_files_format_and_content: .txt files are 2 columns: gene ID and raw counts; Gene IDs correspond to the gene annotation available at https://genome.jgi.doe.gov/portal/pages/dynamicOrganismDownload.jsf?organism=Aspni_NRRL3_1

Submission date

Aug 22, 2018

Last update date

Aug 23, 2019

Contact name

Ronald de Vries

E-mail(s)

fungalphysiology@gmail.com

Phone

+ 31 (0)30 2122600

Organization name

Centre of fungal biodiversity