GSM8712218: mycelium, phenetole, 1 day, flask rep2; Trichoderma cf. simile; RNA-Seq
The mycelium samples were ground in liquid nitrogen, and the RNA was extracted using TRIzol reagent. Residual DNA was removed from the RNA samples by using a DNase treatment. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, USA). The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, and fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of the DNA fragments, adaptors with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments preferentially of 370~420 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. The PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform, and 150 bp paired-end reads were generated.