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Sample GSM2469386

Query DataSets for GSM2469386

Status

Public on Jul 26, 2017

Title

Control strain SAA.111 rep2

Sample type

SRA

Source name

Control strain SAA.111

Organism

Aspergillus nidulans

Characteristics

genotype/variation: control
biological replicate: 2
growth conditions: Mycelium grown in liquid minimal medium at 37°C for 3 days
tissue: Mycelium

Treatment protocol

Gene replacement of ANIA_08444 was performed with the double joint-PCR (DJ-PCR), as described in Yu et al. (2004): the cassette containing the selectable marker argB (ANIA_04409) was inserted to replace the entire locus ANIA_08444.

Growth protocol

The strain used in this study is SAA.111 (genotype veA1; biA1; ΔargB :: trpC; riboB2; pyroA4; wA3) and it was grown according to Bernreiter et al. (2007).
Minimal medium was prepared with 0.4% w/v glucose as C source and ammonium tartrate at final concentration of 10 mM as N source. Trace metal solution 0.02% v/v was supplemented to the medium.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA from mycelia was extracted from 100 mg of finely ground tissue, by using the RNeasy Plant Mini Kit (Qiagen), coupled with the on-column DNaseI digestion. RNA Integrity was measured using the Agilent bioanalyzer and RNA integrity numbers (RINs) were >7 for all the samples. RNA concentrations were measured using a ND-1000 spectrophotometer (NanoDrop). One microgram of extracted RNA was retro-transcribed using the ProtoScript II Reverse Transcriptase (NEB) following the manufacturer’s instructions.
Libraries were prepared from 3µg of total RNA extracted from the mycelium after 3 days of growth at 37°C with the SMARTer Stranded RNA-Seq kit (Clontech). The isolation of mRNAs was performed using the Illumina beads and the TruSeq protocol (Illumina), as described in Behr et al. (2016). The synthesis of cDNA and shearing were performed with ten ng mRNA, according to the manufacturer’s instruction. The enrichment step was carried out using 14 cycles of PCR. The libraries were then checked using a 2100 Bioanalyzer (DNA High sensitivity Kit) to estimate the average fragment size. Library quantification was performed using the KAPA library quantification kit (KAPA Biosystems) and a ViiA7 Real-Time PCR System (Life technologies). The pooled libraries (at a concentration of 20 pM) were sequenced on an Illumina MiSeq (MiSeq reagent kit V3, 150 cycles) generating 76 base pairs paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

Control-2

Data processing

Raw FASTA files were imported in CLC Genomics Workbench 9.0.1. Sequences were filtered and trimmed according to the following criteria: sequence length>35bp, sequence quality score <0.01, no ambiguity in the sequence, trimming using Illumina adaptors, hard trim of 8 base pairs (bp) at the 5’ end and 2 bp at the 3’ end, resulting in a longest length of 64 bp.
For each library, the mapping was performed with the following settings: a maximum hit per reads of 3, similarity fraction=0.9, a length fraction=0.9, a mismatch, insertion and deletion cost of 3 (stringent mapping). The expression values were then calculated using the RPKM method (Mortazavi et al., 2008).
In order to highlight the differentially expressed genes, a t-test with 2 groups (control and celA-) each composed of three biological replicates was performed. Only genes with a p-value below 0.05 were selected. A cut-off threshold was also applied to the fold change (log2 FC absolute value>1).
Genome_build: The A. nidulans FGSC A4 reference transcriptome was downloaded at the Aspergillus Genome Database site http://www.aspergillusgenome.org/download/sequence/A_nidulans_FGSC_A4/current/
Supplementary_files_format_and_content: The tab-delimited text file includes: Transcript IDr, p-value, Gene symbol, Gene annotation, Unique gene reads, RPKM values for each sample, average of the three biological replicates, Gene size.

Submission date

Jan 26, 2017

Last update date

May 15, 2019

Contact name

Gea Guerriero

E-mail(s)

gea.guerriero@list.lu

Phone

+3522758885096

Organization name

Luxembourg Institute of Science and Technology (LIST)