Neb clean and concentrate kit

In-vitro transcribed (IVT) RNA or purified Schizosaccharomyces pombe total RNA was used as spike-ins where noted. The IVT RNA was produced by first amplifying a linear DNA fragment encoding NanoLuc using Q5 polymerase (NEB #M0494S), and purifying the DNA using an NEB clean and concentrate kit. The RNA was then made using a T7 Highscribe kit (NEB #E2040S), treated with DNase I (NEB #M0303L) and purified using an NEB clean and concentrate kit (NEB #T2030).
For the S. pombe RNA, fission yeast (FY527) was grown in YES media (5 g/L yeast extract, 30 g/L glucose, 225 mg/L adenine, histidine, leucine, uracil and lysine hydrochloride) at 32°C until OD₆₀₀ = 0.5, harvested by centrifugation (3 minutes at 2500 g), resuspended in Trizol, and lysed by vortexing with 0.5 mm zirconia glass beads before extracting RNA using Zymo Direct-zol kits (Zymo #R2072).

In-vitro transcribed (IVT) RNA or purified Schizosaccharomyces pombe total RNA was used as spike-ins where noted. The IVT RNA was produced by first amplifying a linear DNA fragment encoding NanoLuc using Q5 polymerase (NEB #M0494S), and purifying the DNA using an NEB clean and concentrate kit. The RNA was then made using a T7 Highscribe kit (NEB #E2040S), treated with DNase I (NEB #M0303L) and purified using an NEB clean and concentrate kit (NEB #T2030).
For the S. pombe RNA, fission yeast (FY527) was grown in YES media (5 g/L yeast extract, 30 g/L glucose, 225 mg/L adenine, histidine, leucine, uracil and lysine hydrochloride) at 32°C until OD₆₀₀ = 0.5, harvested by centrifugation (3 minutes at 2500 g), resuspended in Trizol, and lysed by vortexing with 0.5 mm zirconia glass beads before extracting RNA using Zymo Direct-zol kits (Zymo #R2072).