RNA-Seq of Aspergillus Welwitschiae: biological replicate
Total RNA was extracted using RNeasy Plant Mini Kit from Qiagen reagent following the manufacturers procedure. To analyze the quantity and purity of total RNA, the Bioanalyzer 2100 and the RNA 6000 Nano Lab Chip Kit from Agilent (CA, USA) were used. The RNA samples were assessed, and only those with a RIN (RNA Integrity Number) above 7.0 were selected. For RNA sequencing (RNA-seq) analysis, three biological replicates were then sent to LC Sciences, 2575 West Bellfort Street Suite 270 Houston, TX 77054 USA (https://lcsciences.com/) for further processing, including library construction, and sequencing. Approximately 5 g of total RNA was used for mRNA purification using the Ribo-Zero Gold rRNA Removal Kit from Illumina (cat.MRZG12324, San Diego, USA). The remaining RNA molecules were fragmented into shorter fragments using the NEBNext Magnesium RNA Fragmentation Module (cat.E6150S, USA), which employs divalent cations under high temperatures. The fragmented RNA was then reverse-transcribed using SuperScript II Reverse Transcriptase from Invitrogen to generate cDNA. This cDNA was used to synthesize second-stranded DNAs labeled with U using E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA), and dUTP Solution (Thermo Fisher, cat.R0133, USA). The resulting double-stranded cDNAs were purified and subjected to end repair, dA tailing, adaptor ligation, and DNA fragment enrichment. The final cDNA library had an average insert size of 300  50 bp. Lastly the cDNA library was subjected to paired-end sequencing (2150 bp) on an Illumina Novaseq 6000 platform, following the recommended protocol provided by the vendor.