GSM6598140: Pit_Cre_Deg; Penicillium italicum; RNA-Seq
Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure Poly(A) RNA is purified from plant total RNA(20ug) using poly-T oligo-attached magnetic beads using two rounds of purification. Because of the 3′ cleavage product of the mRNA contains a 5′-monophosphate，the 5' adapters were ligated to the 5' end of the 3′ cleavage product of the mRNA by the RNA ligase. The next step is reverse transcription to make the first strand of cDNA with a 3′-adapter random primer. and size selection was performed with AMPureXP beads.Then the cDNA were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 15cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 200-400 bp. At last, we performed the 50bp single-end sequencing on an Illumina Hiseq 2500 (LC Bio, China) following the vendor's recommended protocol. Degradome sequencing