sRNA-seq of Magnaporthe oryzae: modcl1/2_2nd
To prepare total mycelial RNA, fungal mycelia were incubated in liquid complete medium (CM) at 25 C for 4 days and collected for total mycelial RNA extraction. For preparation of total RNA from conidiation stage, conidiating mycelia were harvested. Briefly, fungal mycelia incubated in CM at 25 C for 4 days were collected and placed on sterile membrane filter laid on V8 agar medium. After 6 days of incubation sealed with parafilm, followed by one day unsealed incubation for aeration, the membrane filter with conidiation stage were collected and subjected to RNA extraction. Total RNA including sRNA was isolated using miRNeasy mini kit (Qiagen, Hilden, Germany) according to manufacturers instructions. The quality and concentration of each sample were checked by Agilent 2100 Bioanalyzer or 2200 TapeStation (Agilent Technologies, CA, USA). For the synthesis of one cDNA library, RNA samples from three independent replicates were pooled and subjected to reverse transcription. cDNA libraries of sRNAs were constructed using TruSeq Small RNA Library Prep Kit (Illumina, CA, USA) and sequenced by Illumina Hiseq2500. For mRNA sequencing, cDNA libraries were constructed using TruSeq RNA Sample Prep Kit (Illumina) and paired-end sequencing of each sample was conducted by Illumina HiSeq2511.
modcl1/2_2nd_sRNA