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Sample GSM3560428

Query DataSets for GSM3560428

Status

Public on Feb 24, 2021

Title

inter respiratory burst rep1

Sample type

SRA

Source name

Phase III compost mycelium

Organism

Agaricus bisporus

Characteristics

strain: A15
time: ~8.5 h after a burst started

Treatment protocol

~7.5 g compost samples from the immediate surrounding of temperature sensors were harvested, wrapped in aluminium foil, immediately frozen in liquid nitrogen (LN), and stored at -80 °C.

Growth protocol

A. bisporus strain A15 rye spawn (8 g) was inoculated in phase II compost (1 kg) (CNC, Milsbeek, the Netherlands) at 25 °C until temperature peaks developed (as measured with a DS18B20 sensor which was placed in the compost). Boxes were overlayed with plastic film with small holes to allow for a high humidity.

Extracted molecule

total RNA

Extraction protocol

Samples were crushed in LN using mortar and pestle and further ground to a fine powder for 1 min using a Tissuelyser II with a LN pre-cooled steel grinding jar (Qiagen, Venlo, Netherlands) at 30 Hz.
Strand-specific mRNA-seq libraries for the Illumina platform were generated and sequenced at BaseClear BV (Leiden, The Netherlands). High-quality total RNA (checked and quantified on a Bioanalyzer (Agilent)) was used as input for the so-called dUTP library preparation method (Parkhomchuk D et al, Nucleic Acid Research 2009; Levin J et al, Nature Methods 2010). Briefly, the mRNA fraction was purified from total RNA by polyA capture, fragmented and subjected to first-strand cDNA synthesis with random hexamers in the presence of Actinomycin D. The second-strand synthesis was perform incorporating dUTP instead of dTTP. Barcoded DNA adapters were ligated to both ends of the double-stranded cDNA and subjected to a PCR amplification. The resultant library was checked on a Bioanalyzer (Agilent) and quantified. The libraries were multiplexed, clustered, and sequenced on an Illumina HiSeq 2500 (HiSeq Rapid SBS v2 chemistry) with a single-read 50 cycles sequencing protocol and indexing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Single-end sequence reads were generated using the Illumina HiSeq2500 system. FASTQ sequence files were generated using bcl2fastq2 version 2.18. Initial quality assessment was based on data passing the Illumina Chastity filtering. Subsequently, reads clipped (up to minimum read length of 50bp. The second quality assessment was based on thecontaining PhiX control signal were removed using an in-house filtering protocol. In addition, reads containing (partial) adapters were remaining reads using the FASTQC quality control tool version 0.11.5. The final quality scores per sample are provided as Enclosure.
HISAT version 2.1.0 (Kim, Langmead, & Salzberg, 2015) was used to align sequence reads to the Agabi_varbisH97_2 version of the A. bisporus H97 genome (E Morin et al., 2012), which was obtained from MycoCosm (Grigoriev et al., 2014).
Cuffdiff (version 2.2.1), which is part of Cufflinks (Trapnell et al., 2010), was used to identify reads mapping to predicted genes and to identify differentially expressed genes. The bias correction method was used while running Cuffdiff (Roberts, Trapnell, Donaghey, Rinn, & Pachter, 2011). Cuffdiff normalizes the expression level of each predicted gene to fragments per kilobase of exon model per million fragments (FPKM).
Genome_build: Agabi_varbisH97_2 version of the A. bisporus H97 genome
Supplementary_files_format_and_content: Cuffdiff output (gene_exp.diff)

Submission date

Jan 11, 2019

Last update date

Feb 24, 2021

Contact name

Robert-Jan Bleichrodt

E-mail(s)

r.bleichrodt@uu.nl

Organization name

Utrecht University

Street address

Padualaan 8