title: GSM7256153: KO Spores Rep 2 [KO2_Spo]; Aspergillus fumigatus; RNA-Seq
library_construction_protocol: Conidia were collected into water with 0.01% Tween 80 and filtered thoroughly to remove hyphal fragments. Spores were pelleted at 1000xg and resuspended in a volume of 200 uL which was added to 1.5mL screwcap tubes containing 1mL QIAzol Lysis Reagent (Qiagen) and 250uL of 0.5 mm zirconia/silica beads and bead beaten for five minutes. RNA was extracted according to manufacture's instruction with an additional phenol:chloroform:isoamylalchohol (24:1:1) extraction step before RNA precipitation. Total RNA was further cleaned up using RNeasy Mini Kit with on-column DNase digestion (Qiagen) Library was prepared using TruSeq library preparation protocol with poly-A mRNA enrichment Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dTTP. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on  Illumina platforms, according to effective library concentration and data amount