GSM5506265: Ribo-seq wild-type DD12 rep1; Neurospora crassa; OTHER
Tissue was harvested in the dark, pressed in between paper towels to remove excess media, and immediately frozen in liquid N2. Ribosomes were pelleted by sucrose-density centrifugation and ribosome-protected mRNAs were isolated from the pelleted cell extracts using the Qiagen miRNeasy Mini Kit (Cat. # 217004). All subsequent steps up to step 46 in the published ribosome profiling protocol (Ingolia et al., 2012) were followed as described. An rRNA depletion step was not performed, but instead after heat-inactivation of CircLigase, 2.0 μL of GlycoBlueTM, 6.0 μL 5 M NaCl, 74 μL of DEPC water, and 150 μL of isopropanol were added to each tube and precipitation was carried out overnight at -80°C. The DNA was pelleted by centrifugation for 30 min at 20,000 g at 4°C. The pellets were washed with 70% ethanol and allowed to air-dry for 10 min. The pellet was then resuspended in 5.0 μL of 10 mM Tris (pH 8.0), and cDNA synthesis using SuperScript™ III Reverse Transcriptase, followed by barcode addition by PCR amplification using Phusion Hot Start High-Fidelity DNA Polymerase were performed as described (101). Sequencing libraries were quantified and checked for quality on a 2100 Bioanalyzer using a DNA high sensitivity chip per the manufacturer's instructions.