GSM4984891: NH4_S3_paired; Oidiodendron maius Zn; RNA-Seq
Fungal tissue was removed from cellulose membrabes with a sterile scalpel, placed in pre-weighed RNase-free 1.5 mL Eppendorf tubes and flash-frozen in liquid nitrogen. Up to 100 mg of flash-frozen fungal tissue was mechanically disrupted by grinding in liquid nitrogen in a sterile mortar and pestle and placed in RNase-free 1.5 mL Eppendorf tubes on ice. To each tube, 700 μL extraction buffer (100 mM Tris-HCl pH 8, 100 mM NaCl, 20 mM Na-EDTA, 0.1% PVP, 1% sodium-lauryl sarcosine, prepared in diethylpyrocarbonate (DEPC) water) and 700 μL acid phenol were added prior to gently inverting the tubes. Tubes were centrifuged for 5 minutes at 14000 rpm (4 °C); all subsequent centrifugation was also performed at 14000 rpm (4 °C). The uppermost phase of the supernatants was transferred to new Eppendorf tubes on ice, to which an equal volume of acid phenol-chloroform-isoamyl alcohol (25:24:1) was added. Tubes were gently inverted several times, then centrifuged for 5 minutes. Supernatants were transferred to new tubes containing an equal volume of chloroform, tubes were gently inverted several times and then centrifuged for 5 minutes. This chloroform extraction and centrifugation was repeated once. Total nucleic acids were precipitated by addition of an equal volume of isopropyl alcohol to the supernatant and gentle inversion. Tubes were incubated for 30 minutes at -80 °C, then centrifuged 30 minutes. Supernatant was discarded and the pellet was resuspended in 500 μL DEPC water. To each tube 500 μL 6 M LiCl was added, then the tubes were gently inverted and kept overnight in ice at 4 °C. Tubes were centrifuged 30 minutes, then the supernatant was discarded and the remaining pellet was washed with 150 μL 70% ethanol (prepared with DEPC water). The tubes were centrifuged for an additional 5 minutes, the supernatant discarded and the pellet was dried on ice in the fume hood. Once completely dry, the pellet was resuspended in 25 μL DEPC water, then the concentration and quality were determined using a NanoDrop ND-1000 UV-visible light spectrophotometer (NanoDrop, Wilmington, DE, USA). Only RNA samples with 260/280 nm wavelength ratio of approximately 2 and 260/230 nm wavelength ratio of approximately 2 were retained. RNA solutions were treated with PerfeCTa® DNase I according to manufacturer's instructions (Quanta Biosciences™, Beverly, MA, USA). cDNA libraries were prepared for sequencing using standard Illumina protocols for MiSeq