 Mycosphaerella populorum growth on wood chips
Double-stranded cDNA was synthesized from the purified polyA+RNA using Superscript Double-Stranded cDNA Synthesis kit (Invitrogen, USA) and random hexamer primers (Invitrogen) at a concentration of 5µM. The cDNA sheared using Covaris E110 (Covaris, USA) for 75 second at “Duty cycle” of 20% and “Intensity” of 5, and 200-300bp fraction was excised from 8% PAGE, and a PET library was prepared by following the pair end library preparation protocol. Briefly, the DNA was subject to end-repair, and phosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4 polynucleotide kinase respectively in a single reaction, and then 3’ A overhangs generation by Klenow fragment (3’ to 5’ exo minus), and ligated to Illumina PE adapters, which contain 5’ T overhangs. The adapter-ligated products were purified, then PCR-amplified with Phusion DNA Polymerase in 10 to 15 cycles using Illumina’s primer set (Illumina). PCR product of desired size range was purified using a 8% PAGE, and the DNA quality was assessed and quantified using an Agilent DNA 1000 series II assay and Quant-iT dsDNA HS Assay Kit using Qubit fluorometer (Invitrogen), then diluted to 8nM. The final concentration was double checked and determined by Quant-iT dsDNA HS Assay again for Illumina Sequencing.