title: RNA-seq of Fungi
design: Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after which theconcentration, quality and integrity were determined using a NanoDrop spectrophotometer(Thermo Scientific). Three micrograms of RNA were used as input material for the RNA samplepreparations. Sequencing libraries were generated according to the following steps. Firstly, mRNAwas purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation wascarried out using divalent cations under elevated temperature in an Illumina proprietaryfragmentation buffer. First strand cDNA was synthesized using random oligonucleotides andSuper Script II. Second strand cDNA synthesis was subsequently performed using DNAPolymerase I and RNase H. Remaining overhangs were converted into blunt ends viaexonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3 ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare forhybridization. To select cDNA fragments of the preferred 400-500 bp in length, the libraryfragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched usingIllumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XPsystem) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100system (Agilent). The sequencing library was then sequenced on NovaSeq 6003 platform(Illumina) by Suzhou PANOMIX Biomedical Tech Co., LTD.
library_name: PC_41