GSM2627475: N5801_dim1-2_polyA_RNA; Neurospora crassa; RNA-Seq
Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC. Mycelia were harvested and added to a tube containing ~300uL glass beads (260-300 micrometer, acid washed [Sigma Aldrich] and resuspended in sdH2O), 350uL NETS buffer (10mM Tris pH 7.5, 300mM NaCl, 1mM EDTA, 0.2% SDS) and 350uL phenol:chloroform:isoamyl alcohol (25:24:1) and lysed by a bead beater for 10 minutes at room temperature. Samples were cleared by centrifugation, and the aqueous (top) phase was added to 650uL cold 100% EtOH.  Samples were precipitated at -20oC, pelleted by centrifugation, washed with 70% EtOH, and resuspended in DEPC-treated dH2O. RNA was treated with DNase (DNA-free™ DNA Removal Kit, Thermo Fisher Scientific), cleaned (Agencourt® RNAclean XP® beads, Beckman Coulter), and RNA-seq libraries were prepared (KAPA Stranded mRNA-seq kit, KAPA Biosystems)