GSM8309039: N3944_delta-dim5_polyA-mRNA-seq_second-rep; Neurospora crassa; RNA-Seq
Cultures were harvested by vacuum filtration, placed in 2.0mL screw-top tubes, and 300µL of acid washed glass beads suspended in dH2O (150-212µm, Sigma Aldrich cat# G1145-10G), 350µL Acid Phenol:chloroform (5:1; ThermoFisher cat# AM9720), and 350µL NETS (300mM NaCl, 1mM EDTA, 10mM Tris pH 7.5, 0.2% SDS) were added. Samples were vortexed for 10 minutes at maximum speed and centrifuged at 4oC for 5 minutes at 13k rpm. The aqueous layer was transferred to two tubes (~250µL each), 650µL of cold 100% EtOH was added, and RNA was precipitated on ice for 10 minutes and pelleted by microcentrifugation at 4oC for 10 minutes at 13k rpm. The pellet was washed with cold 70% EtOH (made with DECP-treated dH2O), the supernatant was removed, and pellets were allowed to air dry for 15 minutes and resuspended in 50µL DEPC-treated dH2O. Insoluble material was removed by centrifugation, and the initial RNA concentration was measured by Nanodrop. Total RNA (10µg) was resuspended in 50µL of DEPC-treated dH2O. Total RNA cleanup was performed by the Zymo RNA clean and concentrator kit (Zymo Research cat# R1013), per the manufacturer's protocol; the 15µL elution was divided into 4µL for Qubit concentration (RNA HS assay kit; ThermoFisher Scientific, cat# Q32852) and TapeStation (Agilent) quality control analysis, while the remaining 11µL was stored at -80oC. Only total RNA samples with high rRNA:mRNA ratios were used for library construction. Polyadenylated messenger RNA (polyA mRNA) was selected with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs [NEB], cat# E7490L) and polyA mRNA sequencing libraries were generated with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, cat# E7760L and E7765L) per the manufacturer's protocols; quadruplicate polyA mRNA-seq replicate libraries were constructed for each strain. All polyA mRNA-seq libraries were Illumina sequenced at the GC3F on a NovaSeq 6000 as SR118 reads.