GSM7991581: mycelium, d_xyr1_2_cel_rep_1; Trichoderma harzianum; RNA-Seq
Total RNA was extracted using the Magnetic Tissue/Cell/Blood Total RNA Kit (Tiangen, China) according to the manufacturer's instructions. RNA purity and quantity were evaluated using a NanoDrop spectrophotometer (Thermo Scientific, USA), and RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). For the library preparation for transcriptome sequencing, a total amount of 1.5 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). To select cDNA fragments of preferentially 200-250 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. The purified PCR products (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The library preparations were sequenced on an Illumina NovaSeq 6000 platform by Beijing Allwegene Technology (China), and paired-end 150 bp reads were generated. About ~40 M raw reads for each sample were generated.