RNA-seq of Penicillium janthinellum
Source of Penicillium janthinellum strain GXCR  The Penicillium janthinellum strain GXCR was isolated by our lab, a strain with higher copper (Cu) resistance, which can resist 30 mM Cu on TYA plate (10 g Bacto-tryptone (Difco), 5 g yeast extract, 5 g NaCI, 2 g sodium gluconate and 20% agar in 1,000 ml water).   Transcriptome sequencing  The conidia of the GXCR were inoculated to 200 mL TY medium (TYA without agar) containing 0.5 and 3 mM Cu, or without external Cu, respectively, and cultured for 3 d at 32 ºC in a shaker at 200 rpm. The mycelial cultures resulting from different conditions were used for extraction of the total RNA, respectively. The total RNA extraction was conducted with TruSeq Stranded Total RNA LT-(with Ribo-ZeroTM Gold)-Set B according to the manufacturer’s protocol. A 5-μg aliquot of the total RNA was captured from each kind of the mycelial culture by Dynabeads Oligo(dT) (Life technologies, USA). The quality and integrity the RNA sample were controlled not by Bioanalyzer 2100 (Agilent, Germany) but by running agarose gel before us, sheared to fragments of ~200bp, and then reverse-transcripted into cDNAs by SuperScriptIII cDNA Synthesis Kit (Life technologies, USA). The cDNAs were end-repaired by ligation of an A base followed by ligating to Illumina sequencing Truseq V2 RNA Adapter which was supplied by the TruSeq RNA LT V2 Sample Prep Kit (Illumina, Sandiego). The cDNAs resulting from the end modification were amplified by 12 thermal cycles of PCR and then used for construction of cDNA library by using TruSeq RNA LT V2 Sample Prep Kit (Illumina, Sandiego). The cDNA libraries were qualified by Qubit 1.0 (Life technologies, USA) and Bioanalyzer 2100 (Agilent, Germany) before sequencing. Sequencing was performed on the Illumina Hiseq 2000 sequencer with TruSeq SBS v3-HS kits as 2×100 bp paired-end strategy. The quality control of the reads was conducted as the criteria of 20 (error rate = 1%) for quality threshold, 5 bp for window size, and 35 bp for length threshold. The N-containing sequences were removed from the reads following the standard of length threshold of 35 bp. The resulting valid reads from all the sequencing samples were collected, forming a valid read collection. A total of 500,000 reads randomly selected from the valid read collection were subjected to the initial homology analysis as the cut-off values of 1E< 10 and the coverage with over 80% against public nucleotide databases to check contamination of ,the reads. Subsequently, the valid reads underwent de novo splicing as the paired-end method by using the Trinity software (trinityrnaseq_r2011-11-26) as the default parameters.