Transcriptome analysis of yeast luc7 mutants

RNA was isolated as previously described (Wang et al. 2020 (link)). Briefly, strains expressing either LUC7-WT or mutants luc7-nΔ31 and luc7-znf2 and with UPF1 deleted were grown overnight in YPD at 30°C. The following morning, the cultures were diluted in fresh YPD to OD₆₀₀ = 0.1 and grown at 30°C for 2 doublings; after which cells were collected and flash frozen in liquid nitrogen. Total RNA was extracted using hot phenol:chloroform followed by ethanol precipitation. Total RNA (40 μg) from each condition was then treated with DNaseI (Invitrogen) to remove genomic DNA. Samples were ribo-depleted using the Ribocop for Yeast kit (Lexogen) and libraries were prepared using the CORALL Total RNA-Seq V2 kit (Lexogen). Barcoding was carried out using the UDI 12 nt Set B1 (Lexogen), and amplification was done using 15 cycles of PCR. Sequencing was performed on a NovaSeq PE150 by Novogene who also carried out sample demultiplexing.